Mobile phase removal

Problems may be encountered in the analysis of thermally labile compounds, as heat is required for mobile-phase removal and for the transfer of analyte from the belt into the source of the mass spectrometer, and highly involatile compounds which cannot be desorbed from the belt, unless FAB is used for ionization. 

After the preparative plate has been developed and the mobile phase removed, the separated compoimds mnst be located prior to their recovery from the plate. Mobile phase removal after development shonld not involve heating if the compound of interest is thermally labile place the plate in a desiccator for vacuum drying. 

This combination depends firstly on the development of the TLC plate followed by mobile phase removal by drying in an appropriate... 

Stopcocks are used to control the flow rate and should be wide bore to prevent plugging with viscous mobile phases. Removable bottoms are used for those separations that require long times. The bottom can be removed and the column packing pushed out with a plunger (CARE this is not as easy as it sounds). The column packing then can be sectioned, and the desired compounds extracted or washed from the inert phase for further use. 

Sample preparation Dialyze 400 xL plasma against 175 p,L acceptor solution through a Cuprophane membrane (15 kDa cut-off) at 37° for 10 min, inject 500 xL acceptor solution (including the portion used for dialysis) onto column A at 0.71 mL/min, elute the contents of column A onto column B with mobile phase, remove column A from circuit and condition it with 1 mL acceptor solution, elute column B with mobile phase and monitor the effluent. Flush acceptor channel with 5 mL acceptor solution and plasma channel with 8 mL acceptor solution containing 25 p-g/mL TYiton X-100. (Acceptor solution contained 5.9 g NaCl, 4.1 g sodium acetate, 0.3 g KCl, and 1.65 g sodium citrate in 1 L water, adjusted to pH 7.4 with citric acid.)... 

Sample preparation Weigh out amount containing 10 mg triamcinolone acetonide, make up to 50 mL with mobile phase. Remove a 2 mL aliquot and dilute it to 10 mL with mobile phase, filter, inject a 20 piL aliquot of the filtrate. 

Sample preparation Inject 10 p.L plasma into MeCN pumped at 0.2 mlVmin so that the precipitated proteins are removed by 0.5 and 0.2 p,m filters in series. Swith the MeCN containing sample into the mobile phase and allow it to pass onto the analytical column, elute the analytical column in the usual way with mobile phase. Remove the filter unit from the circuit and back-hush it to waste with 100 mM sodixun dodecyl sulfate at 2 mL/min, equilibrate filters with MeCN for 5 min before next iiyection. 

Develop the sheet for a distance of 10 cm in a tank saturated with 100 ml of the mobile phase. Remove the sheet and dry it thoroughly prior to spraying. 

Sample preparation Add 150 tiL water and 300 tiL IS in MeCN to 100 p,L plasma, centrifuge, add 450 tiL of the supernatant to a conditioned (unspecified) Advanced Automated Sample Processor C8 SPE cartridge containing 0.5 mL water ( ), wash with 500 p,L MeCN water 25 75, elute the contents directly onto column A in series with column B using mobile phase, remove column A from the circuit, continue to elute column B with mobile phase, monitor the effluent from column B. 

Place the papers, already spotted with hydroxamic acids and hanging from an empty trough, in the chromatogram box and start the pump. After thirty minutes fill the trough with 50 ml of the mobile phase. Remove the paper six hours later, air-dry and spray with 2 per cent ferric chloride solution in 0 0IN hydrochloric acid. 

To minimize the mobile phase s contribution to conductivity, an ion-suppressor column is placed between the analytical column and the detector. This column selectively removes mobile-phase electrolyte ions without removing solute ions, for example, in cation ion-exchange chromatography using a dilute solution of HCl as... 

Another example is the purification of a P-lactam antibiotic, where process-scale reversed-phase separations began to be used around 1983 when suitable, high pressure process-scale equipment became available. A reversed-phase microparticulate (55—105 p.m particle size) C g siUca column, with a mobile phase of aqueous methanol having 0.1 Af ammonium phosphate at pH 5.3, was able to fractionate out impurities not readily removed by hquid—hquid extraction (37). Optimization of the separation resulted in recovery of product at 93% purity and 95% yield. This type of separation differs markedly from protein purification in feed concentration ( i 50 200 g/L for cefonicid vs 1 to 10 g/L for protein), molecular weight of impurities (<5000 compared to 10,000—100,000 for proteins), and throughputs ( i l-2 mg/(g stationary phasemin) compared to 0.01—0.1 mg/(gmin) for proteins). 

After a chromatogram has been developed the TLC plate is removed from the developing chamber and the status quo is fixed by removing the mobile phase remaining in the layer as quickly as possible. This is properly performed in the fume cupboard so as not to contaminate the laboratory with solvent fumes. If possible the TLC plate should be laid horizontally because then as the mobile phase evaporates the separated substances will migrate evenly to the surface where they can be the more readily detected. A fan or hair dryer (hot or cold air stream)... 

Note It is necessary to remove acid mobile phases completely, since the color reaction only occurs in neutral to weakly acid medium. This is often difficult when cellulose layers are employed so that interference can occur. 

Note The background color depends on the pH of the layer, it is, therefore, affected by the efficiency of removal of acidic mobile phase components before staining. 

Note The reagent can be employed for qualitative and quantitative analysis on silica gel and RP layers. Ammonia, amine and acid-containing mobile phases should be completely removed beforehand. Amino phases cannot be employed. The NBD-chloride reagent is not as sensitive as the DOOB reagent (qv.) on RP phases. 

Detection and result The TLC plate was dried in the air for 30 min and heated to 110 °C for 10 min in order to remove the formic acid from the mobile phase, before immersing the chromatogram in the reagent solution for 10 s. 

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