Gradient program

The column used for blood serum analysis was 100 cm long, 1 mm in diameter and packed with RP 18 reversed phase having a particle size of 10 pm. A concave gradient program was used to develop the separation over a period of 45 min. at a flow rate of 50 pl/min. The initial solvent was 75% methanol 25% water and the final solvent was pure methanol. 

It is seen that a high peak capacity was available but, as a gradient program was used, the isocratic peak capacity is not pertinent. The mobile phase program started with a solvent mixture containing 20% v/v of ethyl acetate in n-hexane and ended with pure ethyl acetate. 

GRADSCF is an ab initio gradient program system designed and written by A. Komornicki at Polyatomics Research. 

The plate number in equation (4.56) corresponds to the value when the effective value of the capacity factor (equal to k when the band is at the column midpoint) is equal to the capacity factor in isocratic elution for the same column. The effective value of the capacity factor, k, is simply 1/1.15b. In most cases k, will be large and equation (4.57) is simplified by equating l/k, to zero. The resolution between two adjacent bands in a gradient program, again analogous to isocratic elution, is e q>ressed by equation (4.58)... 

Elution Isocratic Gradient (programmed temperature, chromathermography, programmed flow)... 

Pulay, P. 1979a. An Efficient Ab Initio Gradient Program. Theoret. Chim. Acta (Berl.) 50, 299-312. 

G. Vivo-Truyols, J.R. Torres-Lapasio and M.C. Garcia-Alvarez-Coque, Enhanced calculation of optimal gradient programs in reversed-phase liquid chromatography. J. Chromatogr.A 1018 (2003) 183-196. 

Lou, D.-W., Saito, Y., and Jinno, K. (2006a). Simultaneous LC determination of ginsenosides using a modified extraction procedure and an improved step gradient program. Chroma-tographia 63, 31-37. 

FIGURE 2 Typical chromatograms ofTween-80 in aqueous solutions (0.1-3%,w/w). HPLC method conditions Waters Symmetry columns (5 lm, 4.6xl50mm) were eluted with a mobile phase containing acetonitrile and 0.1% phosphoric acid (gradient program) with UV detection at 220 nm. Injection volume is 20 lL. 

Flow Rate Gradient Program Detection Reagents ... 

The experimental of peptides or proteins depend more on the type of the column than on the gradient program and are constant within % at various gradient times. The variance in the (p is lower as the size of molecules increases. The values of (p are slightly higher with totally porous particles than with columns packed with superficially porous, nonporous particles and monolithic columns [97]. 

FIGURE 10.21 Comparison between experimental and simulated peaks in gradient elution (Langmuir adsorption isotherm). Injected concentration 2.7g/L injected volume 0.163 xL. Continuous line experimental profile dotted line simulation. The shape of the gradient program is also represented. (Reprinted from Marchetti, N. et ah, J. Chromatogr. A, 1079, 162, 2005. With permission from Elsevier.)... 

Run the respective gradient program from time to time without protein to check for impurities. Inject about 2 ml 0.1 N NaOH to remove residual protein and to clean the columns and tubings. 

Table 6.1.3 Gradient program used to control the HPLC gradient system with column switching for blue-fluorescing pterins ... 

Table 6.1.4 Gradient program used to maintain the flow rate of the HPLC...

Fig. 14a c. Protein unfolding influence of incubation time. Gradient elution of papain on a RP 4 column. Mobile phase A 10 m H3P04 (pH 2.2), B 1 -propanol — water (45 55) with 10 mAf H3Pt)4 gradient program 3 % propanol/min. I — injection time, S — start of the gradient. Incubation a = 0 min, b = 30 min, c = 50 min other conditions as in Fig. 13. (From Ref.5) with permission)...

Fig. 15. Memory effect. Gradient elution of 500 pg phosphorylase kinase on a RP 18 column (250 x 4.6 mm dP = 5 pm). Mobile phase A 0.1% TFA in water B 0.08% TFA in acetonitrile gradient program 0 % B (0-8 min), 46 % (9 min) 68 % (24 min), 75% (33 min) flow rate 1 ml/min. The lower chromatogram was obtained upon sample injection the five blank gradients were performed immediately after the initial separation. Molar mass of the subunits a — 132,000 daltons P — 113,000 y - 43,000 5 - 16,680. (From Ref. 66> with permission)...

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