Resolution gradient elution

The resolution of these columns for protein mixtures, however, was comparably poor. The peak capacity for human serum albumin was near 3 during 20 min gradient elution. Improvement has been reached by covalent binding of PEI (M = 400-600) onto a 330 A silica of 5 pm particle size [38], The peak capacities of ovalbumin and 2a -arid glycoprotein were 30-40 (tgradienl = 20 min). Enhanced peak capacity and resolution probably were due to the more diffuse structure of PEI coupled to silane moieties than that of strictly adsorbed on silica and cross-linked (see Sect, 2.2). Other applications of covalently adsorbed PEI are discussed in Sect. 4.1. 

The HPLC system used consisted of a 30 x 2 mm Luna CN column with linear gradient elution employing two mobile phases A and B (A, 90% H2O 10% acetonitrile B, 10% H2O 90% acetonittile) with both phases containing 5 mM ammonium acetate and 0.2% formic acid. The hnear gradient commenced with 50 50 A B increasing to 100% B after 1 min of the analysis this composition was maintained for 1 min before returning to 50 50 A B after 4 min. Positive-ion ionspray (pneumatically assisted electrospray) was used to obtain mass spectra, with the spectrometer operating at a resolution of 5000. 

Jandera, P. and Churacek, J., Gradient elution in liquid chromatography. I. The influence of the composition of the mobile phase on the capacity ratio (retention volume, band width, and resolution) in isocratic elution — theoretical considerations, /. Chromatogr., 91, 207, 1974. 

With isocratic elution and a sample having solutes with a wide range of polarity it is sometimes not possible to achieve the desired resolution in an acceptably short time. It may be possible to improve the chromatogram using gradient elution. A practical example of the development of a gradient is discussed. 

Second-dimension elution time range The second-dimension elution time range must be determined. The flow rate needs to be optimized for maximum resolution and speed. This will establish the performance of the second dimension. The elution time range can be tuned with either gradient elution and/or by flow rate to determine the sampling rate. 

The stationary phase is selected to provide the maximum selectivity. Where possible, the retention factor is adjusted (by varying the mobile phase composition, temperature, or pressure) to an optimum value that generally falls between 2 and 10. Resolution is adversely affected when k 2, while product dilution and separation time increase greatly when k 10. When this is not possible for all feed components and large differences exist among the -values of the different solutes, gradient elution should be considered. 

Carr and Schelling6 state in detail how the reservations many chromatographers still have toward gradient elution no longer hold true ...gradient elution provides an overall faster analysis, narrower peaks and similar resolution of the critical pair compared to isocratic elution without loss in repeatability of retention time, peak area, peak height, or linearity of the calibration curve. ... 

The various separation parameters should be adjusted to provide optimum resolution. These include mobile phase flow rate, stationary phase particle size, gradient elution, and column temperature (using an optional column oven). 

The gradient elution method for HPLC is the method in which the mobile phase composition is changed in some preprogrammed way in the middle of the run. The device that accomplishes this is called the gradient programmer and is placed between the mobile phase reservoir and the pump. It is useful in experiments in which altering the mobile phase composition assists with the resolution of the mixture. 

Buffered mobile phases are used and the proportions of polar solvents (e.g. methanol, tetrahydrofuran) depend upon the type of derivative employed. Gradient elution is required for the resolution of complex mixtures and analysis times are less than 1 h. Ultraviolet, fluorescence or electrochemical detectors are used depending upon the nature of the amino acid derivatives. 

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