Gradient selection

In order to select the elution conditions for a preparative separation, the concentration of polar modifier at the point of desorption is required (see above). As a general rule of thumb, under overload conditions the main component will elute at approximately two-thirds of the concentration of polar modifier observed in the analytical separation. For example, if a main component desorbs from the analytical separation at a concentration of 30% B under overload conditions it will elute at approximately 20% B. 

Time (minutes) Concentration of buffer B (% v/v) Flow rate (cm3/min) 

A generic gradient recommended for preparative separations is to start the elution at a concentration of B that corresponds to a value 10% earlier that the anticipated desorption then apply a change in concentration of B corresponding to 5% over 15 minutes. To exemplify this approach Table 5.2 shows a gradient, applied to an analytical column, used to purify a peptide that is anticipated to desorb at 20% B under overload conditions. 

It is important to note that the preparative separation is carried out at a low flow rate to allow for mass transfer, diffusion and column wall effects discussed previously. 

Attach an analytical column (e.g. 4.6 mm X 250 mm) to the HPLC system and program the equipment to run the chosen preparative gradient. Run a series of separations at column 

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A wide variety of ID and wD NMR techniques are available. In many applications of ID NMR spectroscopy, the modification of the spin Hamiltonian plays an essential role. Standard techniques are double resonance for spin decoupling, multipulse techniques, pulsed-field gradients, selective pulsing, sample spinning, etc. Manipulation of the Hamiltonian requires an external perturbation of the system, which may either be time-independent or time-dependent. Time-independent... 

Figure 25 H2BC spectrum recorded on cyclosporine (left), compared with a standard gradient-selected HMBC (right).

ID-transient NOE experiments employing gradient selection are more robust and therefore are more reliable for measuring dipolar... 

S = 0.0 ppm)) of oximes 44-51 measured for solutions in DMSO-d based on -gradient selected H, N HMBC experiments"... 

Gawinecki and co-workers reported the structural determination of isomeric products from a similar reaction, the condensation of 1,2-diaminobenzenes 197 with 1,2-dicaibonyl compound 196 <99T8475>. The two different regioisomeric quinoxalines 198, which were produced in nearly equal amounts, were distinguished through the use of advanced NMR techniques including 2D z-gradient selected H, N HMBC. 

Fig. 2. Pulse scheme for the gradient-selected, sensitivity-enhanced X/Y se-HSQC experiment as employed for 31P/15N correlation spectroscopy in Ref. 25. 90° and 180° hard pulses are denoted by solid and open bars, respectively. 2 are delays of length 1/(4 /x,y)> and is a short delay of the same length as the gradient pulse (typically rj 1 ms). Pulse phases are x, unless specified. The ratio of gradient pulse strengths is set to G2/Gi = Yy/Yx, and quadrature detection in Fi is achieved by recording every transient twice and changing the sign of G2 in the second scan.

Fig. 5. 500 MHz HRMAS H NMR spectra of FMOC-isoleucine on Wang resin swollen in DMF- 7 and spun at 4 kHz. Spectrum A shows the presence of dissolved phenylalanine peaks, particularly around 3.0 ppm from its methylene protons, and residual protons of the solvent. Application of a diffusion filter using gradients selectively removes these signals in spectrum B. Reproduced with permission from Ref. 50. Copyright 1999 Elsevier.

Because the sum is not equal to zero, we end up with twisted coherence and no signal in the receiver. We call this a gradient-selected experiment because the gradients are being used to specifically refocus coherence in the desired coherence transfer pathway (XH SQC -> 13C SQC) and to reject all others. In Chapter 10, we will develop the idea of coherence order in a more precise manner, and we will see that coherence order can be either positive or negative. 

Disadvantages of Phase Cycling Compared to Gradient Selection... 

Loss of Sensitivity Due to Overselectivity. Gradient selection means that only a single coherence level can be present at the time of each gradient. With phase cycling we apply a mask with holes at regular intervals, so that more than one coherence level... 

Gradient-Selected HSQC with Phase-Sensitive Data Presentation... 

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