Mobile phase preparation filtration

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. 

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. 

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... 

Initially, the antibodies should be purified prior to prepare the immunoaffinity column. Precipitation with ammonium sulfate, ion-exchange chromatography, gel filtration chraoma-tography or affinity chromatography may be employed with the aim of antibody purification. Activated beads which are coated with bacterial proteins A or G may be used as the support material. Some parameters may be changed for the elution of the sample solution for example the ionic conditions of mobile phase may be changed or chaotropic buffers may be used [11]. 

S-FormylthienoP -AJtellurophenc1 A mixture of 5.0 g (20 mmol) of 6/f-thicno[3,2-6]tellurin, 30 ml of pyridine, and 1.1 g (10 mmol) of selenium dioxide is stirred for 0.5 h at 20°. The mixture is then filtered, the filter cake is washed with 5 ml of pyridine, and the washings and filtrate are combined and poured into hydrochloric acid cooled by pieces of ice. The hydrolyzed mixture is extracted with two 50 ml portions of chloroform, the combined chloroform extract is washed with water, dried with anhydrous magnesium sulfate, filtered, and the solvent is distilled off. The residue is chromatographed on preparative TLC plates (silica gel 60 PF 254, Merck) with benzene as the mobile phase to isolate the product (Rf = 0.6) yield 2.1 g (40%) m.p. 122-125°. 

Bis[methoxymethyl]-2,S-diphcnylte1lurophene 430 mg (1 mmol) of 3,4-bis[chloromethyl]-2,5-diphenyl-tellurophene are added to a solution of sodium methoxide prepared from 40 ml of absolute methanol and 200 mg (8.7 mmol) of sodium and the mixture is heated under reflux for 10 h. The mixture is then allowed to cool to 20°, poured into water, and extracted several times with diethyl ether. The combined extracts are dried with anhydrous sodium sulfate, filtered, and the filtrate is evaporated. The residue is chromatographed on silica gel with benzene as the mobile phase to give the product as a light yellow oil yield 350 mg (83%). l,3-Diphenyl-4//,6//-selenophene[3,4-c]tellurophene A solution of disodium selenide, prepared from 1.58 g (20 mmol) of selenium and 0.92 g (40 mmol) of sodium in liquid ammonia, in absolute methanol is saturated with nitrogen. 1.28 g (3 mmol) of 3,4-bis[chloromethyl]-2,5-diphenyltellurophene are added and the mixture is refluxed under nitrogen for 15 h. The mixture is then filtered, the filter cake is dissolved in chloroform in the presence of activated charcoal, filtered, and the product is recrystallized from chloroform/methanol yield 0.79 g (60%) m.p. 220°... 

In the analytical procedure, an accurately measured aliquot of the product is diluted Avith a diluent (normally the mobile phase) and the resulting sample solution is injected into the HPLC. Because the majority of injectable pharmaceuticals are clear solutions, typically a simple dilution step is all that is needed for sample preparation. However, if the parenteral product is an emulsion or a suspension, appropriate steps must be taken to dissolve the product to achieve a clear solution (ultrasonication, filtration, etc.). For the assay procedure, the sample concentration chosen should be such that the peak areas obtained from multiple injections from the same sample are reproducible with minimum variance (<2% relative standard deviation). Peak shape and retention time also play important roles in the precision of the assay. A tailing factor less than 1.5 and a capacity factor less than 10 for the active peak are generally required for a good analytical method. A reference standard solution having the same concentration and using the same diluent as the sample solution is prepared. 

Liu et al. (2003) focused their method paper on the instrumental set-up for on-line capillary LC coupled with MS-MS via a low flow-rate interface, hoping that miniaturization would improve sensitivity for many biomedical applications. The system design and perfonnance were tested on DHEA sulfate and pregnenolone sulfate quantitation from 5 pL of plasma from one male volunteer. Sample preparation was simple - addition of aqueous methanol and of the deuterated analog internal standards, protein precipitation, supernatant filtration through a Cig bed, evaporation and reconstitution in 100 pL of mobile phase, of which 20 pL were injected for LC-MS-MS analysis. The DHEA sulfate concentration found was in agreement with literature values based on GC-MS and LC-MS studies. 

Sample preparation Finely powder half a tablet, add 9 mL mobile phase, sonicate for 20 min, make up to 10 mL with mobile phase, filter (Whatman type 40 and 0.2 p.m MiUipore), inject an aliquot of the filtrate. 

Sample preparation Powder levodopa/carbidopa tablets or contents of capsules, weigh out an amount equivalent to about 100 ng levodopa, add 30 mL 0.1 M HCl, sonicate, make up to 50 mL with 0.1 M HCl, mix, filter (0.45pm), discard first 5 mL filtrate. 10 mL Filtrate + 50 mL 0.5 mg/mL acetaminophen in MeOH mobile phase 75 175, make up to 100 mL with mobile phase, mix, inject a 20 pL aliquot. 

Sample preparation Powder tablets, add 40 mg pyrimethamine, dissolve in 20 mL MeCN, add 40 mL mobile phase, filter (paper), wash filter with mobile phase, make up filtrate to 100 mL with mobile phase. Dilute a 5 mL aliquot to 50 mL with mobile phase, inject a 20 p.L aliquot. 

Sample preparation Pulverize tablets, weigh out amount equivalent to about 500 pg betamethasone, add 10 mL water, sonicate for 15 min, extract three times with 15 mL chloroform n-butanol 95 5. Combine extracts and filter them through 1 g anhydrous sodium sulfate moistened with chloroform n-butanol 95 5. Collect filtrate and dilute it to 50 mL with chloroform n-butanol 95 5. Remove a 1 mL aliquot, add 0.5 mL 40 pM cortisone in mobile phase, mix, inject a 5 pL aliquot. 

Sample preparation Condition a 500 mg Baker-10 CIS SPE cartridge with 10 mL MeOH, 10 mL water, and 10 mL saturated aqueous disodium EDTA. Condition a 500 mg Beiker-10 COOH cartridge with MeOH ethyl acetate 10 90. Dissolve 25 g honey in 50 mL 100 mM pH 4.0 disodium EDTA-Mcllvaine buffer, filter. Add the filtrate to the CIS SPE cartridge, wash with 20 mL water, wash with 400 p.L ethyl acetate, air dry under vacuum for 5 min, elute with 50 mL MeOH ethyl acetate 10 90. Add a 5 mL aliquot to the COOH SPE cartridge, wash with 5 mL MeOH ( ), elute with 10 mL mobile phase, inject a 100 p,L aliquot. 

Sample preparation Crush tablets, weigh out powder equivalent to 50 mg enalapril ma-leate, dissolve in 25 mL mobile phase, filter, dilute filtrate with an equal volume 1 mg/mL lisinopril in mobile phase, iiyect tui ahquot. 

Sample preparation Add 0.1 (rabbit) or 1 (rat, monkey) plasma to 1 mL 100 mM pH 5.0 acetate buffer and 50 jlL Glusulase (from Helix Pomatia, contains 10000 U/mL sulfatase and 90000 U/mL p-glucuronidase, DuPont), heat at 37° for 1 h, cool to room temperature, add 15 mL diethyl ether, shake mechanically at high speed for 10 min, centrifuge at 3033 g for 10 min, freeze in dry ice/acetone. Remove the organic layer and evaporate it to dryness under a stream of nitrogen, reconstitute the residue in 500 j.L mobile phase, filter (0.45 p.m), inject a 150 xL aliquot of the filtrate. 

Sample preparation 150 u,L Plasma + 150 jjlL ethylene glycol water 40 60, mix, filter (5 H,m), filter (0.22 xm) while centrifuging at 4° at 1000 g for 5 min, iiyect a 150 jtL aliquot of the filtrate onto column A and elute to waste with mobile phase A, after 5 min elute column A to waste with mobile phase B, after 8 min direct the effluent from column A onto column B, after 1.5 min remove column A from the circuit, elute column B with mobile phase D, monitor the effluent from column B. deem column A by eluting to waste with mobile phase C for 19.5 min, re-equilibrate column A with mobile phase A for 4 min. 

Sample preparation Condition a Bond-Elut CIS SPE cartridge with 2 column volumes MeOH and 2 column volumes 100 mM pH 6.0 sodium phosphate buffer. 1 mL Serum -l-100 p,L 100 p.g/mL IS in MeOH water 10 90 -I- 2 mL 100 mM pH 6.0 phosphate buffer, mix, add to the SPE cartridge, wash with 1 mL phosphate buffer, wash with 1 mL MeOH phosphate buffer 15 S5, dry under vacuum, elute with 500 (aL MeOH. Evaporate the eluate to dr3mess under a stream of nitrogen at 40°, reconstitute with 200 p,L mobile phase, filter, inject a 50 p,L aliquot of the filtrate. 

Sample preparation Tablets. Pulverize tablets, add MeOH, shake for 20 min, filter, wash solid with MeOH, dilute filtrate with mobile phase, inject a 20 pL aUquot. Urine. 2 mL Urine -1- 2 mL 1 M pH 3.25 KH2PO4 -t- 4 mL ethyl acetate, vortex for 20 min, centrifuge at 734 g for 5 min. Remove the organic layer and evaporate it to dryness under a stream of nitrogen at 40°, reconstitute the residue in 2 mL mobile phase, iiyect a 20 pL ahquot. 

Sample preparation Weigh out capsule contents equivalent to 50 mg ketoprofen, add 80 mL MeOH, sonicate for 10 min, make up to 100 mL with MeOH, filter. Dilute a 4 mL aliquot of the filtrate to 100 mL with mobile phase. Mix a 10 mL aliquot of the diluted solution with 10 mL 50 p,g/mL ibuprofen in mobile phase, make up to 100 mL with mobile phase, iiyect a 50 jrL aliquot. 

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