Buffers gradients

Fig. 3. QAE-Sephadex gradient separation of the B fruit extract. An 18 mg (uronic acid equivalents) sample of extract was dissolved in 20 ml of 125 mM imidazole-HCl buffer (pH 7.0) and applied to the column. The column was then eluted with 50 ml 125 mM buffer followed by a 125 mM to 1.5 M buffer gradient (500ml), and, finally, 50 ml of 1.5 M buffer. Fractions of 5 ml were collected and assayed for uronic acids. Groups of fractions (26-41, 45-50, 53-75 and 84-100) were pooled, concentrated by ultrafiltration and analyzed by HPLC.

Fig. 2.64. Representative chromatogram of tea infusions. Conditions C18 column acetonitrile-aqueous acetate buffer gradient absorbance at 210 nm. Peak identities 1 = epigallocatechin (EGC) 2 = caffeine 3 = epicatechin (EC) 4 = epigallocatechin gallate EGCG) 5 = epicatechin gallate (ECG) 6 = internal standard (naringenin). Reprinted with permission from W. E. Bronner et al. [180].

Under these reducing conditions of hydrolysis of tryptophan peptides, cystine is reduced to cysteine and its coelution with proline using standard buffer gradients, makes quantitation difficult. Thus, cysteine and cystine are generally derivatized prior to acid hydrolysis by oxidation to cysteic acid with performic acid 21 or alkylation, upon reduction in the case of cystine, with iodoacetic acid 21 or, more appropriately, with 4-vmylpyridine)22 23 50 Conversion of cysteine into 5- 3-(4-pyridylethyl)cysteine bears the additional advantage of suppressing epimerization via the thiazoline intermediate, thus allowing for standardization of the acid-hydrolysis dependent racemization of cysteine in synthetic peptides)24 ... 

While salt or buffer gradients at constant pH were employed for elution in all of the above procedures, Vreeman et al. (1977) demonstrated an improved separation of the K-casein components on DEAE-... 

P-Casein (13 mg) containing 0.036 fid M-P-C was subjected to plasmin hydrolysis for 45 min and the reaction mixture was dissolved in 7 mL column buffer (5mM Tris—3mM NaCl—urea, pH 8.55) together with 100 mg whole casein that had been alkylated with iodoacetamide. The sample solution was applied to a column (1.6 X 50 cm) of DEAE-cellulose equilibrated with column buffer. Elution was with a NaCl gradient (3.0-155mM) in column buffer (gradient volume, 1.0 L) 5.0 mL fractions were collected. Under the conditions used as as-caseins remained adsorbed to the column K-caseins were eluted in Fractions 35-56 Am measurement (----------) radioactivity (--) (28). 

Ultrasphere ODS. Mobile phase A 0.05 M SDS, 3% n-propanol, pH 2.5 with phosphate buffer, sodium perchlorate added to balance conductivity with solvent B. Mobile phase B 0.112 M SDS, 3% n-propanol, pH 2.5 with phosphate buffer. Gradient program A to B in 12 min. Peak identification (1) hydroquinone (2) resorcinol (3) catechol (4) phenol (5) p-nitrophenol (6) o-nitrophenol (7) p-chlorophenol (8) p-bromophenol. "Reproduced with permission from ref. 5. Copyright 1984 Elsevier Science Publishers."... 

Set up simple buffer gradient system according to Figure 9.9. 

Table 9.1 Suggested TEAB buffer gradients and retention times for purification of nucleotides by MPLC...

Once the sample is loaded onto the column, start the fraction collector and pump the buffer from the 50 mM buffer to the column at a rate of about 4 mL/min. As this buffer is used up, the higher concentration buffer will be drawn into the flask via the tubing to provide the buffer gradient. 

Hydroxyl apatite chromatography elution via phosphate buffer gradient. Fractions 15-18, 1 ml each. f / r 1 CnA on lonn... 

Figure 9.11. Comparison of the band separation of linear, wedge, and ionic strength gradient gels. The T reaction of a sequencing experiment carried out on M13 mp8 DNA was run on three different 40 cm 6% polyacrylamide gels, using (a) a standard linear gel, (b) a 0.35-1.05-mm wedge gel, and (c) a 0.05 0.50 Af Tris buffer gradient. [Reprinted, with permission, from A. T. Bankier and B. G. Barrell, in Nucleic Acids Sequencing A Practical Approach , C. J. Howe and E. S. Ward, Eds., Oxford University Press, New York, 1989. IRL Press at Oxford University Press 1989.]...

RP-HPLC, conducted with different stationary phases (most often C4- and C-8, but also C18) using aqueous acetonitrile or a phosphate-buffered gradient or iso-cratic conditions are relatively common. 

Phenylureas in MRM 150 ml RW — PLRP-S or C-18 CH3CN/phosphate buffer, gradient elution 72... 

Amino acid analyzer (computer assisted buffer gradient system)... 

The high sensitivity, resolving power, ease, and speed of the method make this an excellent means of critical assessment of purity of amino acid derivatives [88,89]. Typically, elution of derivatives is from a small-diameter (5-10 m, 300 A pore size) reversed phase (RP) support using a linear gradient of acetonitrile in aqueous buffer. Gradients are usually from 30% B to 100% B over 30 min. The following buffer systems are widely used, and it is recommended that at least one be employed for the determination of derivative purity. 

Gel plates Set up buffer gradient gel( see 4-cutter protocol). Prerun gel 40-45W for 30x40x0.04cm gel (should be approx. 1300V). 

Figure 5.29 Separation of a hydrolysate standard on a totally sulfonated cation exchanger. Column 1154110T temperature program 46°C from 0-4 min, 46-70 °C from 4-9 min, 70 °C from 9-32 min, 70-46 °C from 32-33 min eluent sodium citrate buffers gradient linear between pH 3.15,4.25, and 6.40 flow rate 0.60mL/min ...

C eluent sodium citrate buffers gradient step gradient between pFI 2.70 and 6.40 flow... 

Hartwick and Brown [17] successfully separated nucleotide mono-, di-, and triphosphates of adenosine, guanosine, inosine, xanthosine, cytidine, uridine, and thymidine by using a strong anion-exchange column and an acidic phosphate buffer gradient. 

In order to avoid many of the technical difficulties of sedimentation in homogeneous media (convection, associations, etc.), a continuous density gradient is often used. After a sedimentation run in a buffered gradient, established before the run in the centrifuge tube, a hole is punched in the bottom of the tube, and the solution collected drop by drop. Monitoring is by absorption, determination of radioactivity and enzyme activity, etc., and, frequently, a marker of known molecular weight is added to the solution. 

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