Apparatus fraction collectors

Stalcup aiid co-workers [14] adapted this method to a continuous elution mini-prep electrophoresis apparatus shown in Fig. 11-3. In this apparatus, the end of the electrophoretic gel is continuously washed with elution buffer. The eluent can then be monitored using an HPLC detector (Fig. 11-4) and sent to a fraction collector where the purified enantiomers, as well as the chiral additive, may be recovered. In this system, the gel configuration was approximately 100 mm x 7 mm, and was aircooled. The number of theoretical plates obtained for 0.5 mg of piperoxan with this gel was approximately 200. A larger, water-cooled gel was able to handle 15 mg of... 

Figure 4-1. Components of a simple liquid chromatography apparatus. R Reservoir of mobile phase liquid, delivered either by gravity or using a pump. C Glass or plastic column containing stationary phase. F Fraction collector for collecting portions, called fractions, of the eluant liquid in separate test tubes.

The core - flood apparatus is illustrated in Figure 1. The system consists of two positive displacement pumps with their respective metering controls which are connected through 1/8 inch stainless steel tubing to a cross joint and subsequently to the inlet end of a coreholder 35 cm. long and 4 cm. in diameter. Online filters of 7 im size were used to filter the polymer and brine solutions. A bypass line was used to inject a slug of surfactant solution. Two Validyne pressure transducers with appropriate capacity diaphragms are connected to the system. One of these measured differential pressure between the two pressure taps located about one centimeter from either end of the coreholder, and the other recorded the total pressure drop across the core and was directly connected to the inlet line. A two - channel linear strip chart recorder provided a continuous trace of the pressures. An automatic fraction collector was used to collect the effluent fluids. 

Magnesium speciation (Section ni.A) in serum was carried out using an anion exchange column for protein separation, with mobile phase at pH 7.4 the effluent was collected in an automatic fraction collector. On-line quantitation of the protein fractions was carried out by DA-UVD, and Mg determination was carried out from the automatic sampler in a GFAAS apparatus, measuring at 202.8 nm . ... 

Column chromatography apparatus, including a suitable low-pressure pump, online UV monitoring at 280 nm and a fraction collector. 

The main difficulty of the air seal seeems to be the fraction collector, but a capillary channel will hold a drop permanently and let it pass only on the pressure of the next one, so that a permanent seal is achieved. In older types of apparatus the fraction collector stood inside the electrophoretic chamber, but this is of course a serious practical disadvantage. A problem in rigid chambers is the change of pressure due to slight rises in temperature during the run, which pushes the sealing drops out of the fraction collector with loss of water-saturated air, but with a soft plastic sheet such pressure changes are compensated by the hood itself. 

In vitro drug release from transbuccal disc devices was determined at 37°C using a USP dissolution apparatus connected through a microprocessor-controlled peristaltic pump to a fraction collector. The cumulative amount released as a function of time was calculated from the drug concentration data after compensating for the total amount removed in the collected sample fractions. 

Requires the use of specialized HPLC apparatus connected to a fraction collector or an on-line 3-radiodetector. 

Column chromatography apparatus, including a suitable low-pressure pump, on-line UV monitoring at 280 nm and a fraction collector. Chromatography media Sephacryl S-200 (SF), Sephadex G-25 (SF) (Pharmacia). 

Plastic joint chps or Keck clips (Fig. 5.22a) are used for holding ground-glass joints firmly together and may be used to replace clamps and support stands at certain points when building apparatus (see p. 109) and are essential when using rotary evaporators (p. 122). The main weakness of these otherwise useful devices is that they soften at about 130°C and this may allow the joints to separate. Therefore they should never be used at the hot end of a distillation, for example. The clip should be used to hold a distillation adapter on the end of a water condenser, or the flasks onto a fraction collector, but never on the distilling flask or to hold the condenser onto the still head (Fig. 5.22b). 

If only a few milliliters of a liquid are to be distilled, the apparatus shown in Fig. 5 has the advantage of low hold-up—that is, not much liquid is lost wetting the surface area of the apparatus. The fraction collector illustrated is known as a cow. Rotation of the cow about the standard taper joint will allow four fractions to be collected without interrupting the vacuum. 

FIG. 4 Vacuum distillation apparatus with Vigreux column and fraction collector. 

Direct elution techniques involve the collection of the eluate by a continuous stream of buffer, which is then fed to a fraction collector, by way of an ultraviolet monitor if desired. Some examples of successful applications will be given in ch. 9. In principle this type of technique can be scaled up considerably, to the milligram level, but great care in the design of the apparatus is required if the separations are not to be vitiated by heating in the thick gels that are used, and if there is not to be distortion of the zones. Some loss of resolution, as we have remarked, appears inescapable. 

Each separation is initiated by hlling the column with either the upper or lower phase of an equilibrated two-phase solvent system. In order to avoid trapping air bubbles in the column, the solvent should be introduced through the bottom of each column, which is kept in a vertical position. Then, the column assembly is tilted at a desired angle (25°-30°) from the horizontal plane. After the sample solution is introduced into the column, the mobile phase is eluted from the column while the apparatus is rotated at a desired rate (60-80 rpm). In order to retain a large volume of the stationary phase, the lower phase is eluted downward from the upper terminus and the upper phase upward from the lower terminus of the column assembly. The effluent from the outlet of the column is continuously monitored with an ultraviolet (UV) monitor and collected into test tubes using a fraction collector. 

A general description of the apparatus used is given in Fig. 25. The column is thermostatted using a jacket connected to a cooling device. The collector is placed in a cold chamber. Both column and fraction collector can thus be cooled down to —35°C. [This apparatus is quite different from the setup designed by Freed and Sack (1971), where the column is maintained permanently at —42°C with diethylketone.] The various parts of the equipment are described in the following sections. 

Fig. 25. Chromatographic apparatus at low temperatures. T, to the liquid thermostat Ni, nitrogen under controlled pressure PP, peristaltic pump GF, gradient flasks Th, temperature controller (thermocouple) CC, collector control S, security C, cold chamber dd, drop detector FC, fraction collector. Adapted from Balny et al. (1975). Reprinted with permission of Analytical Biochemistry. Copyright by Academic Press.

These essentially consist of a pair of closely spaced, vertical rectangular plates bounded on the sides by the electrodes. The sample and carrier buffer are fed from the top of the slit and travel down in laminar flow to a battery of fraction collectors at the bottom. Unlike the Philpot-Harwell device, which is essentially adiabatic, the thin-film separator can be cooled at the plates. The commercially available device, the Elphor , has a throughput of around 0.1 g/h of protein when operated for multi-component separation. It has been used to separate not only proteins, but cells and other particulate materials. Like the Philpot-Harwell apparatus, it uses a relatively large quantity of carrier buffer and the products are substantially diluted during separation. 

A new recycle isotachophoretic process (93) uses a thin-film geometry with the electrical field perpendicular to the principal flow direction. Leading buffer, a marker dye, feed and trailing buffer are introduced into one end of the slit. An isotachophoretic stack develops perpendicular to flow as the liquid moves downstream. A fraction collector at the outlet collects the fractions, which are recycled until the stack sharpens. A computerized feedback control system keeps the stack centered in the apparatus. It regulates the withdrawal of trailing buffer and the addition of leading buffer in counterflow to the migration of the stack, based on the position of the marker dye front. 

A fraction collector (Figure 15-7) is an apparatus that contains dozens of small test tubes or vials on a moveable rack. Modem collectors can be programmed to collect fractions by volume, counting drops or by time. They can be programmed further to collect only peaks of interest. The one shown can hold up to 174 12-13 mm tubes, and an LCD displays the tube number, drop count, and help messages. 

Check the apparatus and compare it to Figure El6-2. The directions that follow involve a 12.2 mL column and collecting fractions manually. If you have a fraction collector then please use it. Otherwise, place a multiple unit test tube rack filled with 20 10 X 75 mm test tubes at the outlet. 

A typical block scheme of gel chromatographic apparatus is shown in Fig. 4.6.4. The mobile phase flows from the solvent container, C, into degassing unit, D, and through filters, F, reaches the pumping system, P, which transports it via the pulse damper, PD, and the sample injecting system, I, into the column, CO. The effluent from the column enters the detector, DE, and flows through the volumeter, V, into the fraction collector, F. 

The components to be described next are the first and last links in the chain of distillation apparatus. The still pot contains the substance to be distilled and the receiver and the fraction collector take up the purified and fractionated distillate, respectively. 

The fraction collector 23 normally contains sixty 20 ml receiving tubes and is buUt into a vacuum desiccator with a wire-gauze safety cover. It can be employed at atmospheric pressure and at reduced pressures down to 1mm. After 30 tubes have been filled a signal sounds alternatively, the apparatus may be made to switch over automatically to the next circle of tubes. When all the tubes are full there is another signal and the reflux controller is switched off so as to stop the distillate take-off. 

The part of the apparatus between the syphon and the fraction collector can easily be taken apart and is so constructed that the distillate is not contaminated bj grease. 

Countercurrent Chromatography Procedure. The entire column (pair of coiled multilayer columns connected in series) was filled with the stationary phase. The apparatus was then rotated counterclockwise at 600 rpm in planetary motion while the mobile phase was pumped into the inlet of the column at a flow-rate of 2.2 mL/min (head to tail elution mode). Maximum pressure at the outlet of the pump measured 80 psi. After a 1-hour equilibration period, the sample was loaded into the Rheodyne injector loop and injected. Effluent from the outlet of the column was continuously monitored with a Shimadzu UVD-114 detector at 312 nm and fractions collected with a Gilson FC-lOO fraction collector to obtain approximately 8.8 mL of eluant in each tube (during a 4-min interval). Retention of the stationary phase was estimated to be 930 mL (74%) by measuring the volume of stationary phase eluted from the column before the effluent changed to mobile phase (330 mL) and subtracting this volume from the total column capacity of 1260 mL. 

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