Page 520 - Tandem Techniques

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closely spaced fractions to be collected with minimal cross-contamination and dilution. Sample
recoveries are better than 80% and virtually independent of the nature of the sample. A diagram of the
fraction collector is shown in Figure 13.15. The coaxial capillary interface (to the fraction collector)
was made from a 1/16 in. Swagelok T, and a 2 cm length of 700 µm O.D. and 530 µm I.D. fused silica
tubing. Fluid was continuously pumped through the T by a mechanically actuated syringe, at a flow-rate
of about 100 µl/ min. If the coaxial sheath flow was dispensed with the capillary interface was could be
rendered conductive by coating it with gold-filled epoxy resin, and curing for 15 minutes at 120°C.

The electrophoretic separations were carried out on capillary tubes, 50 cm long, 52 to 75 µm I.D.,
having a detection window for the UV detector situated 20 cm from the end. The activity of the
capillary walls was reduced by derivatization with (3-aminopropyl)triethoxysilane. Samples were
introduced both by electro-kinetic and/or hydrodynamic procedures. During fraction collection, the disk
was moved by a stepping motor as each peak was eluted. The separation was monitored by a variable
wavelength UV detector at either 200 or 254 nm, and besides monitoring the separation, the output was
used to actuate the disk, and move it on, to allow the next peak to be deposited onto a clean portion of
the disk. The time taken for the solute to pass from the detector cell to the end of the capillary was taken
into account in the fraction collector program. The eluates were collected sequentially on the perimeter
of the disk, and after each peak was eluted, the same portion of disk was replaced under the capillary
tube exit to collect between-peak eluent, or any zones that were not specifically required.

The MALDI TOF-MS instrument was that manufactured by Laser/Tec Research and was equipped with
a neodymium-YAG laser. Under typical conditions, a spot of 50 µm diameter was sampled with 30 µJ
pulses. All spectra were measured in the linear time-of-flight mode (1.3 m flight path). The ions were
accelerated by a potential of about 30 kV, and the resulting ion current monitored at 2 ns intervals.
Spectra were generated from about 12 to 75 single consecutive acquisitions with 2-10 point