Mobile phase filtration

Eluate evaporation and redissolution in mobile phase, filtration. 

Once suitable ionization conditions have been established, LC separation can be optimized. As with any LC system, attention needs to be paid to column choice, correct tubing diameters, zero dead volume connections, use of guard columns, and mobile phase filtration and de-gassing. Modem LC pumps can deliver reliable gradients at low flow rates but for capillary LC, precolumn flow splitting or specialized pumps may be necessary. Mobile phase composition and pH should be chosen to... 

Mobile-phase degassing is an important step in the LC/MS experiment and can be accomplished via on-line membrane or vacuum devices, sonication, helium sparging or as part of the mobile-phase filtration step. Degassing will eliminate pump cavitation, ensure reproducible retention times and minimize possible sputtering from the ion source. 

Solid phase extraction with the Sep-Pak CIS cartridges, evaporation of the eluent to dryness, dissolving the residue in mobile phase, filtration using 0.45 pm filters... 

In contrast to vapour phase chromatography, the mobile phase in liquid chromatography is a liquid. In general, there are four main types of liquid chromatography adsorption, partition, ion-chromatography, and gel filtration. 

Figure 4-2. Size-exclusion chromatography. A A mixture of large molecules (diamonds) and small molecules (circles) are applied to the top of a gel filtration column. B Upon entering the column, the small molecules enter pores in the stationary phase matrix from which the large molecules are excluded. C As the mobile phase flows down the column, the large, excluded molecules flow with it while the small molecules, which are temporarily sheltered from the flow when inside the pores, lag farther and farther behind.

The metabolic and pharmacokinetic profile of sucralose (this is a novel intense sweetener with a potency about 600 times that of sucrose) in human volunteers was studied by Roberts and coworkers [82]. Part of this study was realized using PLC in the following chromatographic system in which the stationary phase was silica gel and the mobile phase was ethyl acetate-methanol-water-concentrated ammonia (60 20 10 2, v/v). Separated substances were scraped off separately, suspended in methanol, and analyzed by filtration, scintillation counting, or enzymatic assay. It was shown that the characteristics of sucralose include poor absorption, rapid elimination, limited conjugative metabolism of the fraction absorbed, and lack of bio-accumulative potential. 

Acetochlor and its metabolites are extracted from plant and animal materials with aqueous acetonitrile. After filtration and evaporation of the solvent, the extracted residue is hydrolyzed with base, and the hydrolysis products, EMA and HEMA (Figure 1), are steam distilled into dilute acid. The distillate is adjusted to a basic pH, and EMA and HEMA are extracted with dichloromethane. EMA and HEMA are partitioned into aqueous-methanolic HCl solution. Following separation from dichloromethane, additional methanol is added, and HEMA is converted to methylated HEMA (MEMA) over 12 h. The pH of the sample solution is adjusted to the range of the HPLC mobile phase, and EMA and MEMA are separated by reversed phase HPLC and quantitated using electrochemical detection. 

In SEC analysis of additive extracts from polymers, the effect of the extraction solvent on the mobile phase is less critical than in HPLC analysis. The extraction solvents typically employed generally do not interfere with the SEC mobile phases. Moreover, the same solvents are often used both as extraction solvent and as mobile phase. Therefore, there is no need to evaporate the extract to dryness prior to analysis and then to redissolve it in a suitable solvent. Typical extraction procedures often produce extracts that generally contain a small amount of wax. Frequently, removal of such oligomers from an extract is necessary, e.g. by means of precipitation, centrifuging, precolumn filtration or protection (use of a reversed-phase guard column). In SEC separations the presence of polyolefin wax does not usually disturb provided that the MW of the wax is higher than that of the analysed compounds. 

HPLC allows a quantitative determination with relatively simple extractions. In many cases, extraction only involves a heating of the commodity with water, followed by filtration and injection onto an HPLC column. In the determination of caffeine, theobromine, and theophylline in cocoa, coffee, or tea, as well as in other foods, there is scarcely a month that passes without a new paper on this assay. Kreiser and Martin provide typical conditions for analysis.28 In their studies, samples were extracted in boiling water and filtered prior to injection onto the HPLC column. The HPLC conditions used a Bondapak reversed phase column and a mobile phase of water methanol acetic acid (74 25 1) with detection at 280 nm. This method is accurate, precise, and conserves time. It has also been adopted by the AOAC as an official method for the determination of theobromine and caffeine in cocoa beans and chocolate products.29... 

Bio-Sil TSK- 250 gel filtration column (7.5mm x 30cm) was obtained from Bio-Rad (Richmond, CA), and used with 0.2 M phosphate (pH 4) mobile phase. 

Collect the precipitated product by filtration and redissolve it in 400 ml of benzene. Repeat steps 4-5 several times to assure complete removal of unreacted TsT. The residual TsT may be detected by HPLC using a 250 X 3.2 mm LiChrosorb (5 pm particle size) column from E. Merck. The separation is done using a mobile phase of hexane, and peaks are detected with a UV detector. 

For oxidative detection removal of dissolved air from the mobile phase is necessary to prevent air bubble formation at the column outlet, which disturbs the electrolysis process. Vacuum filtration usually is sufficient to remove enough air for bubble-free operation. However, air readily redissolves in the mobile phase. Continuous helium sparging is therefore the only effective degassing method for longer periods. 

Left, Charlie Focht of the Nebraska State Agriculture Laboratory prepares the mobile phase for an atrazine assay. Note that the vacuum flask is positioned in an ultrasonic cleaner bath. Simultaneous vacuum filtration and sonication provide a more efficient means for degassing. Right, Charlie adjusts the flow rate setting on the HPLC pump. 

The problem is solved by prefiltering all mobile phases and samples before beginning the experiment. For mobile phases and large sample volumes, this involves utilizing a vacuum apparatus that draws the liquid through a 0.5-pim filter. Since such filtration involves a vacuum, the mobile phase is automatically degassed as well, so the filtration need not be a separate step. An efficient operation would be to filter a mobile phase with a vacuum apparatus while simultaneously sonicating. See Workplace Scene 13.1. 

Distinguish between gel permeation chromatography and gel filtration chromatography in terms of mobile phases that are used and application. 

Prepare 500 mL of a mobile phase that is 25 mM KC1, 5 mM MgCl2, and 50 mM tris-(hydroxym-ethyljamino methane (TRIS or THAM). Adjust the pH to 7.2 using concentrated HC1. Filter and degas this mobile phase using a vacuum filtration apparatus equipped with 0.45-/LL filters. 

Mobile phases and samples contain small particulates that can damage the column and other hardware unless they are removed by filtration. 

Membrane absorbers are continuous chromatographic supports, which circumvent some of the above-mentioned problems of particulate stationary phases. They were originally derived from membrane (filtration) technology. The immobilization of interactive (ionic, hydrophobic, or biospecific) groups on the surface of microfiltration membranes was found to increase the selectivity of certain separation procedure. Ideally such activated membranes, or membrane adsorbers, allow the selective adsorption of certain substances and substance classes, which may subsequently be eluted by means of a stepwise change of the mobile phase (elution buffer). More complete information on the various types of modern membrane technology can be found in some recent reviews [e.g., 31-33]. 

Because of the diaphoretic and laxative effects, the composition of Sambuci flos (Sambucus negra L., black elder) has been extensively investigated by TLC. Samples for TLC analysis were preparated by refluxing 1.0 g of air-dried, powdered flowers of Sambucus negra with 10 ml of methanol for 30 min. The suspension was filtered, and the filtrate was concentrated and redissolved in 5 ml of methanol. Separation was performed on silica layers using 10 different mobile phases 1 = ethyl acetate-formic acid-acetic acid-water (100 11 11 27, v/v) 2 = ethyl acetate-formic acid-water (8 1 1, v/v) 3 = ethyl acetate-formic acid-water (88 6 6, v/v) 4 = ethyl acetate-methyl-ethyl ketone-formic acid-water (50 30 10 10, v/v) 5 = ethyl acetate-methyl-ethyl ketone-formic acid-water (60 15 3 2, v/v) 6 = ethyl acetate-formic acid-acetic acid-methyl-ethyl... 

Another HPTLC method has been developed for the separation of kaempferol and quercetin in the extract of Ginkgo biloba leaves showing beneficial effect in brain diseases. Leaves were refluxed with methanol for 30 min then filtered. The filtrate was refluxed with 25 per cent HC1 for 60 min then neutralized with ammonia and the clear supernatant was applied for HPTLC. Silica plates were predeveloped in chloroform-methanol (1 1, v/v). Separation was performed with toluene-acetone-methanol-formic acid (46 8 5 1, v/v) as the mobile phase using incremental multiple development. A densitogram illustrating the good separation charactersitics of the system is shown in Fig. 2.42. The relative standard deviation (RSD) of the method was low (1.37 and 1.40 for kaempferol and quercetin,... 

FIGURE 1.25 HPLC determination of impurities in a levothyroxin (L-T4) formulation. Experimental conditions Column, Chiralpak QN-AX (150 rum x 4 rum ID) mobile phase, acetonitrile-50 mM ammonium acetate (60 40, v/v) (pHa 4.5) flow rate, 0.7 mLmiu UV detection, 240 nm temperature, 25 C. Sample, T4-200 tablets (Uni-Pharma, Greece) containing 0.2 mg L-T4 sodium per tablet the tablet was pulverized, suspended in methanol-10 mM sodium hydroxide (1 1 v/v) and after ultrasonication for 5 min the residues were removed by filtration. An aliquot of 10 xL of the filtrate was directly injected. (Reproduced from H. Gika et al., J. Chromatogr. B, 800 193 (2004). With permission.)... 

Fill to volume with mobile phase A (20 mM ammonium acetate buffer at pH 3.75) and filter an aliquot of the extract through a Whatman GDX PTFE membrane filter into an HPLC vial after discarding the first 2mL of the filtrate. 

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