Column semi-preparative

Saponification extraction using a Chem Elut column, semi-preparative HPLC (Lichrosorb Si 60, 250mmx4.0mm, 5pm)... 

Columns used for size-exclusion chromatographic separations of macromolecules with different molecular masses are generally longer (25-100 cm) and broader (6-10 mm) than conventional analytical columns. Semi-preparative and preparative columns have internal diameters from 0.6 to 5 cm and even larger columns are used for industrial pilot-plant and process separations (see Chapter 6). 

There are two types of sample valve commonly used in LC, the internal loop valve and the external loop valve. In order to improve the seating and eliminate leaks, the valve faces are sometimes made from appropriate ceramics. The internal loop valves are largely used with small bore columns, that is to say columns having internal diameters of less than 1.5 mm. The external loop valves are used for larger diameter columns up to semi-preparative columns. 

An open column packed with neutral aluminium oxide (grade III) slurry is generally used for semi-preparative separation of large amounts of carotenoid extract, revealing three broad bands (1) carotenes and epoxy-carotenes constitute the first fraction to elute with petroleum ether, (2) monohydroxy and keto-carotenoids with 50 to 80% diethyl ether in petroleum ether are next, and (3) finally, the polyhydroxy carotenoids elute with 2 to 5% diethyl ether in ethanol or... 

An additional problem exists in which impurities in the displacer itself complicate separation.54 Also, the displacer itself must be removed from the column, which lengthens regeneration time and can adversely affect throughput. Ironically, while the difficulties involved in identifying displacers and in column regeneration have retarded use of displacement as a preparative method, there has been renewed interest in using displacement chromatography in analytical and semi-preparative applications for enrichment of trace compounds.55 56... 

Fig. 7. Semi-Preparative Anion Exchange Purification of a 16-mer Oligodeoxynucleotide on a CIM DEAE Disk Monolithic Column. Conditions Column 0.34 ml CIM DEAE Disk (3X12 mm ID) Instrumentation Gradient HPLC system with extra low dead volume mixing chamber Sample 16mer oligodeoxynucleotide from the reaction mixture - bold line, standards of 1,2,3,4,5,6,7,9,10,11,12,14,15,16mer- thin line Injection Volume 20 pL Mobile Phase Buffer A 20 mM Tris-HCl, pH 8.5 Buffer B Buffer A+ 1 M NaCl Gradient as shown in the Figure Flow Rate 4 ml/min Detection UV at 260 nm...

Another method used a semi-preparative ODS column for the prefractionation of wine anthocyanins. Alcohol from wine samples was evaporated at ambient temperature, the aquoeus rest was centrifuged, filtered and applied into an ODS column (230 X 15 mm i.d.). The column was washed with water, then anthocyanins were eluted with the following... 

RP-HPLC methods have been frequently applied for the investigation of various chemical, biochemical and biophysical processes in in vitro model systems. Thus, the separation of new compounds achieved by enzymatic oxidation of phloridzin was carried out by semi-preparative RP-HPLC. Phloridzin was incubated with a polyphenol oxidase prepared from apple pulp for 6h at 30°C under air agitation. After incubation the suspension was filtered, stabilized by NaF and injected into the RP-HPLC column using diluted acetic acid-ACN gradient. The new compounds were isolated and identified by NMR and MA techniques. The proposed mechanism of the formation of new phloridzin derivatives 3 and 4 is shown in Fig. 2.159. The results illustrate that RP-HPLC can be successfully used for the study of enzymatic processes in model systems [331],... 

After removal of the protein by ultrafiltration, the reaction mixture was subjected to semi-preparative HPLC. Purification was performed using a /iBondapak NH2 column (4.0 mm X 150 mm) with the following conditions CH3CN/H20,5 mL min , 25 °C injection volume 10 pL, 4 mg. 

The major cause of peak asymmetry in LC is sample overload and this occurs mostly in preparative and semi preparative LC. There are two forms of sample overload, volume overload and mass overload. Volume overload results from too large a volume of sample being placed on the column and this effect will be discussed later. It will be seen that volume over load does not, in itself, produce asymmetric peaks unless accompanied by mass overload. Mass overload which, as discussed above, is accompanied by a distortion of the normally linear isotherm, can cause very significant peak asymmetry and, in fact, seriously impair the resolution obtained from the column. 

A 25-mL round-bottom flask was charged with l-[Val-Thr(Bzl)-Val-Thr(Bzl)-DMEDA]2 (180 mg, 102 pmol) dissolved in MeOH (2 mL) and formic acid (approx. 3 mL). The flask was flushed with N2 and 10% Pd/C (102 mg) was added. The N2 atmosphere was then replaced with an atmosphere of H2. The reaction was monitored by C18 RP-HPLC. After 4-5 h, the MeOH was removed under reduced pressure and the thick, heterogeneous soln was diluted with H20 (10 mL). The pH of the soln was adjusted to approx. 7 and the catalyst filtered using a 0.22-pm acetate membrane. The soln was analyzed by analytical C18 RP-HPLC and purified using semi-preparative C18 RP-HPLC. The MeCN in the column fractions was removed under reduced pressure and the sample lyophilized to afford the side-chain deprotected peptidomimetic 3 yield 58 mg (45%) ESI-MS rn/z- [M+H]+ calcd. 1253.5 found 1254.0. 

The TH peptide was purified initially by preparative RP-HPLC on a Rainin AutoPrep System with a Vydac 218TP152022 C18 column (15-20-pm particle size, 300-A pore size, 250 x 22 mm) at a flow rate of 5.0 mL -min The elution gradient was 0-100% B in lOOmin, where A was 0.1% TFA in H20 and B was 0.1% TFA in MeCN, with detection at 229 nm. 1-min fractions were collected, with 10 pL from each fraction used for analytical RP-HPLC. Selected fractions were further purified by semi-preparative RP-HPLC on the same system equipped with a Vydac 219TP510 diphenyl column (5-pm particle size, 300-A pore size, 250 x 10mm) at a flow rate of 2.0mL-min The elution gradient was 0-70% B in 70min, where A was 0.1% TFA in H20 and B was 0.1% TFA in MeCN, with detection at 229 nm. 30-s fractions were collected, with 10 pL from each fraction used for RP-HPLC peptide analyses.1851... 

Separation of these spiroacetal isomers was initially achieved using normal phase semi-preparative HPLC, which allowed re-equilibration of the undesired spiroacetal 35 and thereby a method to convert essentially all of the material to the desired isomer 36 after several cycles. The scale-up of this procedure was greatly facilitated by the determination of a simple method for separating the spiroacetal isomers by flash column... 

In addition to requiring significant bulk material, the timeframe to complete the isolation is considerable. If the maximum analytical load for a 4.6 mm x 150 mm column has been determined to be 5 mg, assuming the isolation will be performed using semi-preparative chromatography (20 mm x 300 mm column), approximately 190 mg of sample can be loaded onto the preparative column. For a 0.1% level unknown, this translates to 190 pg of unknown injected onto the preparative column. Therefore, a total of 27 injections are required. If the assay time were estimated to be 1 hr, it would take at least 27 hr to perform the injections needed to obtain 5 mg (once again assuming 100% recovery). This timeframe does not include the time needed for method scale-up development, concentration and... 

Elute the peptides with a linear gradient of acetonitrile in acidified water at a flow rate between 0.8 and 1.3 mL/min depending of the internal diameter of the column. Flow rate can be 0.8-1 mL/min if the column has 4.6 mm of internal diameter and increased to 1.3 mL/min for a semi-preparative column (7 mm of... 

Analytical and semi-preparative reversed-phase columns (e.g. Waters Symmetry and Delta-Pak Ci8 Millipore). 

These semi-preparative methods are useful where identification is required but for quantitative and comparative analytical purposes much more rapid sampling techniques, such as automated headspace and solid phase microextraction (SPME), may be preferred. Both of these techniques give similar results for most volatiles. In the former, the vapour above a heated sample is removed by a syringe or gas flushing and injected onto a GC column, either directly or after trapping on a suitable absorbent and thermal desorption. In SPME, the vapour is absorbed on to a suitable bonded medium on a special needle and then injected into the gas chromatogram. 

Peptide synthesis reagents and materials Use NovaSyn TGR resin (Novabiochem) for C-terminal amide synthesis and Fmoc amino acid monomers (Novabiochem) including Fmoc-Arg(Pbf)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Gln(Trt)-OH, Finoc-Glu(OfBu)-OH, Fmoc-His(Trt)-OH, Fmoc-Lys(Boc)-OH, and Fmoc-Trp(Boc)-OH. Other peptide synthesis reagents are as for PNA (above) with the addition of 1,2-ethanedithiol (EDT, >98%, Fluka). A Phenomenex Jupiter C-18 column (analytical and semi-preparative) may be used for reversed-phase HPLC. 

Analyze the crude product and purify by reversed-phase HPLC using an analytical or semi-preparative column, as appropriate, heated to 45° C. Monitor by UV at 260 nm. Buffer A 0.1% TFA (aq.), Buffer B 90% acetonitrile + 10% Buffer A (v/v). 

Semi-preparative reversed-phase HPLC column was a Waters p.bondapak C18 column 7.8 x 300 mm (Waters, UK, Ref. 84176). 

Fig. 5.10. Semi-preparative purification of 4-hydroxynonenal dinitrophenylhydrazone in microsomal fraction from 0.5 g rat liver. Reversed-phase column, Lichrosorb Si60 (51 m) solvent dichloromethane (50% in water flowrate 1.5 ml/min detection 340 nm injection 100-200 /il (a) Standard (b) Liver microsomal fraction. (Benedetti et...

---