Linear gradient

The most commonly used gradients are linear gradients where the starting solvent is gradually mixed with a second gradient-forming solvent at the column entrance to yield a volume fraction

mobile phase modulator that increases huearly with time ... 

TABLE 16-14 Expressions for Predictions of Chromatographic Peak Properties in Linear Gradient Elution Chromatography under Trace Conditions with a Small Feed Injection and Inlet Gradient Described by op = opo + pt (Adapted from Refs. A and B). 

Fig. 3-2. Semipreparative RP-HPLC profile of eyelo(Arg-Lys-X-Pro-X-Ala). The erude sublibrary (160 mol) was dissolved in 0.1 % (v/v) TFA and applied to a Whatman Partisil 10 pm ODS-2 (1 x 50 em) eolumn. The peaks were eluted using a 40-min linear gradient of 0-25 % aeetonitrile in water at a flowrate of 7 mL min . Fractions were collected every 2 min and pooled in three fractions as indicated by arrows 130 pmol of peptides was reeovered (yield 81 %). (Reprinted with permission from ref. [75]. Copyright 1998, Ameriean Chemieal Soeiety.)...

Anion-exchange chromatography on a column of TSK DEAE-650 M (EM Science) in 30% methanol. Elution with a linear gradient of NaCl concentration from 0 to 1M. 

Fig. 3. Cation-exchange chromatography of protein standards. Column poly(aspartic acid) Vydac (10 pm), 20 x 0.46 cm. Sample 25 pi containing 12.5 pg of ovalbumin and 25 pg each of the other proteins in the weak buffer. Flow rate 1 ml/min. Weak buffer 0.05 mol/1 potassium phosphate, pH 6.0. Strong buffer same +0.6 mol/1 sodium chloride Elution 80-min linear gradient, 0-100% strong buffer. Peaks a = ovalbumin, b = bacitracin, c = myoglobin, d = chymotrypsinogen A, e = cytochrom C (reduced), / = ribonuclease A, g = cytochrome C (oxidised), h = lysozyme. The cytochrome C peaks were identified by oxidation with potassium ferricyanide and reduction with sodium dithionite [47]...

Figure 5.6 Positive-ion electrospray spectrum obtained from the major component in the LC-MS analysis of a purified recombinant 62 kDa protein using a Cig microbore 50 X 1 mm column and a flow rate of 50 p.lmin . The starting buffer (buffer A ) was 0.1% TEA in water, while the gradient buffer (buffer B ) consisted of 0.1% TEA in acetonitrile-water (9 1 vol/vol). The running conditions consisted of 0% B for 5 min, followed by a linear gradient of 100% B for 55 min. Reprinted from J. Chromatogr., B, 685, McAtee, C. P., Zhang, Y., Yarbough, P. O., Fuerst, T. R., Stone, K. L., Samander, S. and Williams, K. R., Purification and characterization of a recombinant hepatitis E protein vaccine candidate by liquid chromatography-mass spectrometry , 91-104, Copyright (1996), with permission from Elsevier Science.

The HPLC system comprised a 75 ftm x 15 cm PepMap column with a linear gradient of acetonitrile/0.1% aqueous formic acid (5 to 50% acetonitrile over 45 min) at a flow rate of 250 nlmin . Positive-ion electrospray ionization was employed using a nanospray interface. MS-MS specna were acquired over the range m/z 40 to 2000 at a rate of 1 s per scan. 

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. 

The Dionex system uses a Garbo Pac PA-1 anion exchange column and a CarboPac PA-1 Guard. The column was loaded with 25 pi of the RG solution and eluted with a linear gradient of 0 - 0.5 M NaOAc in 0.1 N NaOH during 50 minutes. The flow rate was 1.0 ml/min and the process was monitored using a PE detector. 

Compounds that were included in the pharmacologic profile of [ H]MDA binding were subjected to reverse-phase HPLC analysis to assess their relative lipophilicity. Briefly, each compound (10 pg) was injected onto a Waters Nova-Pak C18 column and eluted with a linear gradient from 95 percent buffer A 5 percent buffer B to 20 percent buffer A 80 percent buffer B (buffer A=95 percent water, 5 percent acetonitrile, 0.1 percent ammonium acetate buffer B=20 percent water, 80 percent acetonitrile,... 

Initial conditions 90 10 Linear gradient 30 70 in 7 min Re-equilibrate for approximately 3 min Divert flow to waste for approximately 3 min after injection 100... 

Increase pH by 0.2 units/t, linear gradient (cation exdiange) pH varied from 2-6 in 20t,... 

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