Fraction collection

Fraction collection of fragments from a 100-bp DNA ladder was also achieved in a PDMS chip [664] or a glass chip. In the latter example, a small reversed field was maintained in the separation column to halt or slow down later migrating DNA in order to assist collection of a DNA fraction [665]. In one report, a single peak from the IEF separation channel has been isolated and transferred to a subsequent channel by means of microfluidic valve control [449], 

FIGURE 6.38 (a) Schematic operation showing DNA bands migrating through the intersection formed by the separation and side channels, (b) Schematic operation showing the target DNA captured by the electrode in the side channel [96]. Reprinted with permission from Elsevier Science. 

4 Fraction Collection A component of a fractional distillation assembly critical for the satisfactory operation of the fractionation column is the fraction cutter. Its purpose is to allow collection of the distillate and to control the reflux ratio. A fraction cutter should be designed so that no major disturbance of column equilibrium occurs when a change of receivers is made. A widely used manually operated fraction cutter is shown in Fig. 2-16 this is a typical setup for fractional distillation. Two alternative fraction cutters are shown in Figs. 2-17 and 2-18. 

For high precision distillations, it is advantageous to have an automatically controlled fraction collecter. Although these are more expensive, they allow precise control of the reflux ratio and require less attention than their manually operated counterparts. They usually consist of a timing device and a solenoid-controlled valve that is opened automatically on occasion to collect some distillate. The reflux ratio is controlled by the ratio of time the valve spends in the closed and opened positions. In view of the time saved and the greater control available from the use of an automatically controlled fraction cutter, their purchase ( 100) is highly recommended. 

The line between the detector and fraction collector must have a low volume in comparison with the mobile phase flow rate. 

The line (PFTE tube) between the detector cell and collecting flask is 20 cm x 0.3 mm i.d. Not more than 1 s should elapse between the recorded signal and outflow of the sample zone, thus ensuring that fraction collection can be made correctly. What should the minimum mobile phase flow rate be  

Minimum flow rate = I4mm s = 840rnrn rnin = 0.84 rnl rnin  

For occasional preparative work, fractions may be collected in flasks or glass reaction vessels at the detector outlet. If a fraction collector is used, this should be 

The figures in Section 2.4 give more information on the purity of poorly resolved peaks. However, in the case of mass overload these drawings are no longer valid because the peaks are triangular and in fact influence each other. The profiles of touching peaks can be unfavourable so that the second peak cannot be obtained as a pure fraction, in contrast to the first one.  

The purity of the fraction obtained decreases if the peaks are insufficiently resolved (Fig. 20.4). This situation can be improved by rejecting the middle fraction, which can then be recycled if necessary (see Section 20.5). 

As already mentioned, it is a prerequisite for preparative HPLC that the mobile phase is of the highest purity in order to guarantee the purity of the collected fractions after removal of the solvent. 

After the column the eluent and purified product mixture is depressurized violently and heated to about 20°C to vaporize the eluent. Theoretically it is easy to separate two phases by simple decantation. However, the practice is much different the liquid (or solid) pure product forms a mist (or a dust) that is taken away from the traps by the gas flow and special devices must be used to stop the liquid (or solid) in the traps. The most efficient devices used are a patented model of cyclonic separators. The eluent turns rapidly around the axis of a cylindric tube. The heavy phase carried by the gas is centrifugated and agglomerates on the walls of the tube before falling to the bottom of the tube. The gas phase is evacuated by the top of the tube. 

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Methyl 2-hydroxy-2-carbomathoxy-4-haptenoate (3), A solution of dimethyl mesoxalate 2 (1.46 g, 10 mmol] and 1-pentene 2 (0 70 g, 10 mmol) in CH2CI2 was heated at 140 C lor 16 h. The solvent was reirroved and the residue distilled under reduced pressure. The fraction collected between 90 and 105°C (0 5 torr) was diluted with Et20 (20 mL), washed with water and dned The residue after evaporation of the solvent, gave on dislillation 1 55 g of 3 (62%), bp 89-90 C (0 2 torr). 

Also purified by adding 2g NaBH4 to 1.5L butanol, gently bubbling with argon and refluxing for 1 day at 50°. Then added 2g of freshly cut sodium (washed with butanol) and refluxed for 1 day. Distd and the middle fraction collected [Jou and Freeman J Phys Chem 81 909 I977. ... 

In a 2-1. three-necked, round-bottomed flask fitted with a liquid-sealed mechanical stirrer, a dropping funnel, and an efficient reflux condenser are placed 720 g. (226 cc., 4.5 moles) of bromine (Note i) and 1.5 g. of sulfur (Note 2). A glass tube is connected to the top of the condenser to carry the evolved hydrogen bromide to a gas trap (Org. Syn. 14, 2). Sixty-nine grams (69 cc., 0.52 mole) of dry paraldehyde (Note r) is added slowly, with stirring, over a period of about four hours. The reaction proceeds under its own heat during the addition of the paraldehyde subsequently the mixture is heated externally for two hours at 60-80°. The solution is distilled and a fraction collected over the range 155-175° (Note 3). 

S. Blomherg and J. Roeraade, Preparative capillary gas cliromatography. IF Fraction collection on rtaps coated with a very tliick-film of immobilized stationary phase , J. Chromatogr. 394 443-453 (1987). 

Q. L. Xie, K. E. Markides and M. L. Lee, Supercritical fluid extraction-supercritical fluid clrromatogi aphy with fraction collection for sensitive analytes , 7. Chromatogr. Sci. 27 365-370(1989). 

Fig. 3-9. Preparative HPLC of 100 mg of the test racemate 8 in a single 2 mL injection using a 250 x 4.6 mm i.d. column containing (5)-Glu-(5)-Leu-DNB CSP. Conditions mobile phase ethyl acetate, flowrate 2.0 mL min , UV detection at 380 nm. Injection 2 mL of 50 mg mL racemate solution. Fractions collected before and after the indicated cut point were 98.4 % ee and 97 % ee pure, respectively. (Reprinted with permission from ref. [86]. Copyright 1999, American Chemical Society.)...

Packed column SFC has also been applied to preparative-scale separations [42], In comparison to preparative LC, SFC offers reduced solvent consumption and easier product recovery [43]. Whatley [44] described the preparative-scale resolution of potassium channel blockers. Increased resolution in SFC improved peak symmetry and allowed higher sample throughput when compared to LC. The enhanced resolution obtained in SFC also increases the enantiomeric purity of the fractions collected. Currently, the major obstacle to widespread use of preparative SFC has been the cost and complexity of the instrumentation. 

The /3-chloropropionic acid is best isolated by direct distillation under reduced pressure (Note 1) using a 250-cc. modified Claiscn flask (Org. Syn. 1, 40). The nitric acid is collected up to ioo°/2omm. using a water condenser. The condenser is then removed and the residual /3-chloropropionic acid is fractionated, collecting as pure product the portion boiling at 105-107720mm. The yield is 3i 3i.5g. (S4—55 Per cent of the theoretical amount) (Note 2). The product solidifies on cooling and melts at 41 41.5°. 

To obtain the trimethylene chlorohydrin, the distillate from this operation is heated for about one hour on a steam bath in order to drive out most of the excess hydrogen chloride. The distillate is then fractionated under reduced pressure (Note 3) in a modified Claisen flask (Org. Syn. 1, 40). The fractionating side arm should be 25 cm. in length. The fractions collected under 10 mm. are to 55°, 55-57°, 57-65°, 65-85°, 85-105°, residue. 

Trimethylene chlorohydrin cannot be distilled under atmospheric pressure without some decomposition. The fractionation can be carried out at ordinary pressures when the fractions collected are up to 1250, 125-158°, 158-164°, 164-190°, 190-210° and residue. This procedure is less desirable as some hydrogen chloride is evolved and the product turns dark on standing. 

The colorimetric procedure has been applied to each of the fractions isolated from the partition column. The response to the color test has allowed an accurate prediction of the general type of infrared spectra ultimately found. The color test has also been applied to fractions collected from the gas chromatograph. Of the major responses observed when the pyrethrum mixture is passed through the chromatograph, three of the components respond to the color test. At least two other pyrethrin-like compounds of long retention and small quantity also give the color test. No infrared data are available on these. 

Figures 2 through 9 are infrared spectra of fractions collected from partition columns, gas chromatography, thin-layer chromatography, or a combination of these separation techniques. Figure 10 is the infrared spectrum of a compound isolated by gas chromatography after hydrolysis of a pyrethrum concentrate. In this case the compound is a long-chain ester. All the infrared spectra were made with a Perkin-Elmer Model 221 instrument. The following operating parameters were used. A liquid demountable cell with a 0.01-mm path length was employed.

When this oil was distilled in vac (18mm), the fraction collected between 28° and 34° corresponded ahnost exactly to the oxo-ozonide, CsHgO, d 1.070g/cc at 22/22° RI 1.3798 at 22°. It expld on heating with great violence. 

Once the boiling point starts to rise, it goes up quite rapidly. The fractions collected between 75° and 90° contain a little product and can be reworked if a second distillation is carried out. 

The direct linking of HPLC to mass spectrometry removes the need for fraction collection and with it the potential problems discussed above. It does, however, introduce a number of other problems which have been mentioned in earlier chapters of this book. 

Cross-flow ultrafdtration equipment.—The device used is shown in Figure 1. It included a glass reactor (R) with temperature, pH and stirring control, a Minitan pump (P) (Millipore, Bedford, USA), a Harp hollow fiber membrane cartridge (M) (Romicon-Supelco, Bellefonte, USA) with a cut-off of 2000 daltons, and a permeate exit (f) for fraction collection. The retentate (r) was returned to the reactor. 

The filtration effidency is commonly expressed by the fractional collection effidency T(x), see Eq. (3.2.1), rather than by considering the total mass, as the filtration process depends strongly on the size x of the particles entrained in the carrier gas [1]. 

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