Preparative chromatography fraction collectors

Experimental studies to construct a binding curve will be described at pH 4.5. To prepare the gel filtration column, obtain a chromatography column (about 1.5 X 15 cm). It should be equipped with a porous glass disk and a stopcock at the bottom to control flow rate. Clamp the column to a ring stand and connect the bottom tubing to a fraction collector containing 50 test tubes. If... 

Preparative liquid chromatography and fraction collectors are widely used to purify target compounds for applications found in many disciplines ... 

After the mobile phase exits the column, possibly carrying analytes, it will almost always go through one or more detectors (the only exception likely would be that the mobile phase might be collected in a fraction collector as might be done in preparative chromatography). Detection is the topic of the next section. 

Fraction collectors allow automatic collection of the effluent and are the first to be applied in preparative gel chromatography. The shift of the collecting vials is controlled by the impulses from the device responding to the change of their weight, from the volumeter, from the timer or from the peak-slope detector. 

Low-pressure chromatography typically requires hours, so a fraction collector is a necessity. It is best to configure the system so that a tick mark is made on the strip chart recorder during fraction changes. Fractions can be analyzed by analytical HPLC to determine which ones contain the peptide of interest. Fractions are then pooled and lyophilized. In cases in which preparative HPLC is the next step, the pooled fractions can be pumped directly onto the column. 

A variety of fraction collectors are available from a number of manufacturers and include those which are able to collect low microlitre volumes in addition to those used in preparative HPLC which can collect much larger volumes. Fraction collectors which are to be used with HPLC systems require very rapid tube changing facilities and should also be resistant to the solvents used with HPLC systems thus, fraction collectors which are designed for use with low pressure chromatography are usually inadequate. A major feature in the more recently marketed fraction collectors has been the introduction of advanced microprocessor control which provides a number of capabilities ranging from simple timed collection to total integrated control of the whole HPLC system. 

Safety devices such as air sensors and pressure transducers are built into preparative HPLC units, together with a series of valves. These devices create dead volume and contribute to the extra column volume. A large-scale chromatography unit is composed of valves for selection of buffers and feed solutions, at least two pumps, the separation column, and, in most cases, at least one detector. Instead of a fraction collector, a combination of valves is often used. These sources of dead volume create typical washout kinetics, which contribute exponentially to the band-broadening processes. For the industrial scale, the equipment is mainly customer designed. For medium scale, modular units are available [51]. Attention should be paid to extra column volume when systems are compared. Extra column effects are an important parameter of the quality of a system and should be considered when a system is purchased. 

HPLC equipment dedicated to high-osmotic-pressure chromatography is used for the fractionation of narrow polymer fractions from broad distribution samples. This technique, which employs columns that are packed with a control pore glass of very narrow pore distribution, separates polymers by molecular weight as a function of osmotic pressure. When this approach is coupled with a fraction collector the technique can generate polymer fractions in significant quantities for further study by nuclear magnetic resonance, (NMR), FTIR, or other spectroscopic techniques. This technique can offer superior resolution to the previously mentioned preparative GPC. This technique has been applied to the characterization of both copolymers and homopolymers. 

Another classification of TLC is off-line versus on-line (Nyiredy, 1992). Classical TLC is off-line in that the different steps are performed separately. Overpressured and rotational preparative planar chromatography can be performed on-line by use of an injector, flow-through detector, and fraction collector. 

RPC involves the use of centrifugal force to accelerate the flow of solvent from the feed-point at the center to the periphery of a rotating plate. Up to 72 samples can be separated and quantified in situ by analytical RPC. One sample is applied as a circle for micropreparative and preparative RPC, for which separations can be carried out off-line or on-line with elution from the layer and recovery in a fraction collector (48). A variety of N- and S-type chambers with the prefix designations N (normal), M (micro), U (ultramicro), and C (column) are used for RPC, differing mostly in the size of the vapor space (153). Nyiredy (45) has described commercial instruments (Chromatotron and Rotachrom) and the various modes of RPC and covers preparative layer chromatography, including RPC, in Chapter 11 of this Handbook. 

The main difference between the first four methods [normal chamber RPC (N-RPC), microchamber RPC (M-RPC), ultra-microchamber RPC (U-RPC), and column RPC (C-RPC) 1 lies in the size of the vapor space, that is an essential criterion in rotation planar chromatography. The circular mode of development is always used for on-line preparative separations by all of these methods. The sample is applied near the center of the layer, and the mobile phase is forced through the stationary phase from the center to the outside of the round plate/planar column. The separated compounds are eluted from the layer/planar column as a result of the centrifugal force and collected in a fraction collector. 

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