Gradient former

HPLC has also been used to determine the residue levels of dinifroaniline herbicides. Pendimethalin was quantified by HPLC under the following conditions apparatus, Spectroflow 400 solvent delivery system. Model 430 gradient former, and Kratos Model 783 with UV absorbance detection at 239 nm column, Cig reversed-phase (25cmx 3.0-mm i.d.) temperature, 40 °C mobile phase, acetonitrile-water (7 3, v/v) flow rate, 1 mL min . ... 

Where binary, ternary or quaternary gradient elution (p. 91) is required, a microprocessor controlled low-pressure gradient former is the most suitable (Figure 4.31(c)). The solvents from separate reservoirs are fed to a mixing chamber via a multiport valve, the operation of which is preprogrammed via the microprocessor, and the mixed solvent is then pumped to the column. For the best reproducibility of solvent gradients small volume pumps (< 100 gl) are essential. 

Place solution I and II in the gradient former (ISCO Model 570, Lincoln, NE, or Advantech GR-40, Tokyo, Japan). 

A systematic study has been conducted on the effect of temperature, pH, and salt used in the gradient on resolution (440). Best resolution was observed at 43 , pH 6.8 with KCl or NaCl as the gradient former for the fragments of pRZ2 DNA which is recombinant DNA containing 46(k) base pairs. 

Gradient former for two solutions with different concentrations of a denaturing agent, with a volume of 15 - 20 ml for each solution. The gradient former should be positioned approximately 50 cm over the workplace. 

Prepare a gradient former by placing a stirrer into cylinder 1, which contains both the connection to the second cylinder and the outlet make sure both valves are closed. 

Open the connection between the cylinders (observe flow in cylinder 1). Open the exit valve and slowly pour the gel (over approximately 5 min). Control the flow, particularly when the gradient former is emptying. 

Fig. 10.17. Capillary electrochromatography of PTH-amino acids with gradient elution. Column, 207 (127) mm x 50 pm i.d. packed with 3.5 pm Zorbax ODS particles, 80 A pores. Starting eluent (A), 5 mM phosphate, pH 7.55, 30% acetonitrile gradient former (B), 5 mM phosphate, pH 7.55, 60% acetonitrile flow-rate (through inlet reservoir), 0.1 ml/min gradient, 0-100% B in 20 min voltage 10 kV current, 1 pA temperature, 25°C UV detection at 210 nm electrokinetic injection, 0.5 s, 1 kV. Peaks in order of elution formamide PTH-asparagine PTH-glutamine PTH-threonine PTH-glycine PTH-alanine PTH-tyrosine PTH-valine PTH-proline PTH-tryptophan PTH-phenyialanine PTH-isoleucine PTH-leucine. The concentration of the PTH-amino acids dissolved in the mobile phase was 30-60 pg/ml. Reprinted with permission from Huber et al. [68]. Copyright 1997 American Chemical Society.

Figure 4.26(c). A low-pressure gradient former with three solvents... 

Figure 4-16. Block diagram of HPLC equipment. A typical HPLC set-up consists of a column linked via a pump and gradient former to the solvent reservoirs. The sample application port is located in a separate spur. The column is...

A gradient former is not required for step-wise elution procedures, but most oligonucleotide fractions need gradient elution to achieve the required resolution. That is, the ionic strength, or other property, of the eluting buffer is varied continuously as the elution proceeds. The simplest type of gradient former is made from two open tall... 

Fig. 4.2. Block diagram showing bufler flow from gradient former through pump, column, and monitor(s) to fraction collector and electrical connections from monitor(s) and fraction collector to recorder. The mains power supply to the pump and other accessories may be obtained from the fraction collector mains output which is automatically cut off when a pre-set number of fractions has been collected if the fraction collector has these facilities.

All components except the ammonium persulfate are dissolved and the solution is degassed. Ammonium persulfate is added last, always as the solid. The mixer of the gradient former is then started and the gel solution drained very slowly at first, into the packed plastic... 

The equipment necessary for HPLC analysis includes column, pump, solvent reservoirs, a gradient former, and a suitable detector. It is beyond the scope of this review to cover the rapid, recent developments of HPLC instrumentation. 

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