Sodium HPLC columns

Electrostatic effects have long been recognized in commercial HPLC columns for SEC of proteins (15,21,22). The usual remedy is to add 100 mM salt to the mobile phase. This works here too the Lys and Asp peaks collapse into the Gly peak with 100 mM salt (Eig. 8.8). High concentrations of sodium sulfate were added to determine the role played in SEC by hydrophobic interactions (sodium sulfate, a structure-forming salt, strengthens such interactions). Sodium sulfate increased the retention only of the most hydrophobic amino acids to any extent, and then only when the concentration approached 1 M. Clearly, hydrophobic interaction cannot account for the elution order of amino acids on PolyHEA. 

Purity was confirmed by gel-filtration using a HPLC column packed with Asahipak GS-520HQ and elution with 100 mM sodium phosphate buffer containing 300 mM sodium chloride (pH 6.7). The content of total protein, total sugars, uronic acids, sulfates, nucleic acids, phosphate or fatty acids was assayed by the BCA [32] and Lowry method [33], the phenol-sulfuric acid method [34], the Blumenkrantz method [35], nephelometry [36], absorption at 260 nm, the Bartlett method [37] and the GLC method after methyl-esterification [38], respectively. 

Peptide 41 (500 nmol) was dissolved in 0.1 M sodium phosphate buffer (2mL, pH 7.0). NaI04, dissolved in H20 (0.2 M soln, 13 pL, 3 equiv per Ser residue), was added to the soln. After 5 min, the mixture was applied to a RP-HPLC column for purification, followed by lyophilization of peptide 42. 

Method for catecholamines by HPLC. A few microlitres of a 10 3-Af solution of dopamine, norepinephrine or similar compounds are transferred to a small test-tube and diluted to 20 jul with 0.05 M sodium phosphate (pH 8) at 4 °C [ 102]. 10 jul of a 0.2% solution of fluorescamine in acetone are then added with vigorous shaking. An aliquot portion of this mixture is applied directly to the HPLC column. The derivatives are separated on Hitachi 3011 or 3010-OH gels (column, 50 cm X 3 mm) with methanol-0.05 M Tris-hydrochloric acid buffer of pH 8 (7 3) at room temperature at a flow-rate of 0.72 ml/min. The separation of fluorescamine derivatives of dopamine and norepinephrine with this system is shown in Fig.4.52. 

Fig. 2. (continued) Panel E shows the chromatogram when the BCA-1 in D was run on analytical ion-exchange (sulfonic acid derivatized HPLC column). The gradient is 0 to 1 M sodium chloride in 0.05M K2P04 pH 6.0 and 10% acetonitrile. As is typical the peak is not as sharp as with RP-HPLC but nevertheless only one peak is apparent suggesting that the material in D is pure. 

The Step 2 product was packed into an HPLC column and IM solution pentaethylene hexamine and 0. IM acetic acid in anhydrous ethanol injected into the column. After 2 hours a 0.6M sodium borohydride solution in anhydrous ethanol was injected into the column. After an additional hour unreacted reagents were flushed from the column. The resulting polypentaethylene hexamine silica was able to hold about 800 pmol copper per gram of silica gel. 

Figure 5.9 Enzymatic activities of fractions following HPLC chromatography. A partially purified preparation was fractionated by ion-exchange HPLC (AX-300) with a mobile phase of 0.1 M potassium phosphate. Proteins were eluted with a gradient of sodium acetate. Column eluent was monitored at 280 nm. Fractions were collected, and each fraction was assayed for three different activities.

The reaction mixture contained the substrate ATP, MnCl2, and a sodium acetate buffer at pH 6.0. Reactions were started by the addition of enzyme, and incubations were at 30°C. At intervals samples were withdrawn and injected onto the HPLC column for analysis. Chromatograms obtained showed... 

Activity was assayed in a reaction mixture (1.2 mL final volume) containing the substrate and buffer. The reaction was started by the addition of the enzyme and, after incubation at 37°C for 45 minutes was terminated by the addition of a 15 mAf sodium azide solution. Samples were removed and injected directly onto the HPLC column for analysis. To generate products to be used as standards, oxidation of each of the amines to the corresponding aminochrome was carried out by incubation of the substrate with 30 mAf potassium hexacyanoferrate(IH) solution for 1 hour at room temperature. 

The reaction mixture contained in a final volume of 350 /u.L 30 /umol of Tris-HCl (pH 8), 0.9 /xmol of ATP, 3 /nmol of magnesium sulfate, 6 /u.mol of sodium fluoride, and 50 /nL (2.5 U) of inorganic pyrophosphatase. The reaction was initiated by addition of 50 fiL of enzyme solution. Reactions were terminated by removing aliquots, transferring them to glass test tubes, and capping and heating to 155°C in a sand bath for 5 minutes. Precipitated protein was removed by filtration before injection of 50 fiL of filtrate onto the HPLC column. 

It is most often prepared by acid hydrolysis of sodium silicate followed by emulsification in an alcohol water mixture and subsequent condensation to give solid silica gel. This is then washed and dried for use as HPLC column packing. The exact conditions under which these procedures are carried out (e.g. pH, catalysts, temperature) will affect the properties of the resulting material. The most important qualities with regard to the chromatographic performance of the gel are the average particle size, the particle shape, the specific surface area and the pore size. Other factors which are also important are the pH of the gel surface, the number of active silanol groups and the presence of metal ions. 

Ion - pair HPLC column was employed to separate a synthetic mixture containing lincomycin, lincomycin B, clindamycin B, 7 - epicilindamycin and clindamycin (Figure VIII) (30). Rl detection was utilized with the mobile phase consisting of a 60/40 ratio methanol -water, 2 ml acetic acid per litre ( 0.035 p ), and 0.005 M, DL-10 sodium camphor sulfonate adjusted to pH 6.0 using a 30 cm X 3.9 mm ID prepacked with C p Bondpack column at a flow rate of 1.0 ml/min. Detection by UV can be employed at 214 nm for an additional sensitivity utilizing a mobile phase composed of a 60 40 ratio methanol I water, 0.01 M phosphate buffer, and 0.005 M sodium pentane sulfonate. 

Isolation of Metabolites from Poultry Bile for Spectral Identification. Pooled samples of bile from poultry receiving feed medicated at 25 ppm semduramicin sodium for 7 days were extracted with ethyl acetate followed by chloroform. Abundant metabolites were isolated by collection of fractions from repeated injections on a normal phase silica HPLC column. The recovered fractions were analyzed by Fast Atom Bombardment Mass Spectrometry and proton NMR spectroscopy. 

Urine Saturate citric acid-preserved sample with sodium bicarbonate cleanup on Extrelut cartridge extract with methylene chloride evaporate redissolve in methylene chloride elute from HPLC column with isooctane (or hexane)-isopropanol-methanol HPLC/PCD <1 Atg/L 85-86 Ducos et al. 1985... 

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