Buffer gradient formation

Figure 1 Gradient formation in microfiuidic device monitored with FiTC-dextran. in this device, a constant 1 jil/m flow of 100 ig/ml FITC-dextran (MW 10,000) in Hank s buffer from one syringe and a flow at the same rate of buffer alone was driven by a single-, dual-syringe pump. The fluorescent images were taken at different locations along the main channel. The intensity of the fluorescence was quantitated across the channel for each image and is shown in the plots. Adapted from Walker et al. (2005).

In resting muscle the high concentration of ADP does not decrease the proton gradient effectively and the high membrane potential slows electron transport. ADP, formed when ATP is hydrolyzed by myosin ATPase during contraction, may stimulate electron transport. However, the concentration of ATP (largely as its Mg salt) is buffered by its readily reversible formation from creatine phosphate catalyzed in the intermembrane space, and in other cell compartments, by the various isoenzymes of creatine kinase (reviewed by Walliman et al., 1992). 

Diluted in neutraUzed water, separation on CIS Novapak using gradient elution using methanol and phosphate buffers at pH 7 Formation of ion pairs with cetylpyridinium chloride (CeCl) in water and extraction in butanol, separation on C8 Spherisorb using gradient of acetonitrile, methanol, CeCl/phosphate buffer... 

The comparison of I —> N and N —> I may also be explained by the buffered pH in the diffusion layer and leads to an interesting comparison between a process under kinetic control versus one under thermodynamic control. Because the bulk solution in process N —> I favors formation of the ionized species, a much larger quantity of drug could be dissolved in the N —> I solvent if the dissolution process were allowed to reach equilibrium. However, the dissolution rate will be controlled by the solubility in the diffusion layer accordingly, faster dissolution of the salt in the buffered diffusion layer (process I—>N) would be expected. In comparing N—>1 and N —> N, or I —> N and I —> I, the pH of the diffusion layer is identical in each set, and the differences in dissolution rate must be explained either by the size of the diffusion layer or by the concentration gradient of drug between the diffusion and the bulk solution. It is probably safe to assume that a diffusion layer at a different pH than that of the bulk solution is thinner than a diffusion layer at the same pH because of the acid-base interaction at the interface. In addition, when the bulk solution is at a different pH than that of the diffusion layer, the bulk solution will act as a sink and Cg can be eliminated from Eqs. (1), (3), and (4). Both a decrease in the h and Cg terms in Eqs. (1), (3), and (4) favor faster dissolution in processes N —> I and I —> N as opposed to N —> N and I —> I, respectively. 

In an off-line configuration, a complex peptide mixture from a proteomic sample is loaded onto a SCX column and fractions collected (Fig. 11.1). After the collection of fractions, they are then loaded into an autosampler and analyzed via the traditional RP/ MS/MS approach. Using this system, a variety of buffers and elution conditions may be used (Table 11.1). For example, one may use a volatile salt such as ammonium formate (Adkins et al., 2002 Blonder et al., 2004 Fujii et al., 2004 Yu et al., 2004 Qian et al., 2005a and b) or ammonium acetate (Cutillas et al., 2003 Coldham and Woodward, 2004), collect SCX fractions, lyophilize, resuspend in low acetonitrile and acid, and then directly analyze via RP/MS/MS. In most of the cases, when ammonium acetate or ammonium formate are used, a 20-minute wash period is used to remove the ammonium acetate or ammonium formate prior to the reversed-phase gradient (Table 11.1). However, because fractions are collected and can be buffer exchanged,... 

FIGURE 16 Comparison of a HILIC separation (top) and a reversed-phase separation (bottom). Peak I morphine, peak 2 morphine 3- glucuronide.Top column Atlantis HILIC Silica, 4.6x50mm, 3.0 lm gradient from 90% to 50% acetonitrile with lOmM ammonium formate buffer, pH 3.0 flow rate 2.0mL/min. Bottom Atlantis dC g, 4.6x50mm, 3.0pm mobile phase 2% acetonitrile with lOmM ammonium formate buffer, pH 3.0 flow rate l.4mL/min. 

If the second dialysis step against external buffer is omitted during the formation of LUV, transmembrane pH gradients can be formed by running... 

Recently, Titier et al. [76] were able to reach LOQs of 2 ng/mL in blood by applying solvent extraction, RP C18 separation under gradient mode (acetonitrile/ammonium formate buffer 4mmol/L, pH 3.2), and ESI(-t)-MS-MS SRM mode to the determination of different tricyclic and MAQI anti depressants and their metabolites. 

To HEPES buffer (100 mL, 200 mM, pH 7.5) were added ManNAc 15 (1.44 g, 6 mmol), PEP sodium salt (1.88 g, 8 mmol), pyruvic acid sodium salt (1.32 g, 12 mmol), CMP (0.64 g, 2 mmol), ATP (11 mg, 0.02 mmol), pyruvate kinase (300 U), myokinase (750 U), inorganic pyrophosphatase (3 U), /V-acctylneuraminic acid aldolase (100 U), and CMP-sialic acid synthetase (1.6 U). The reaction mixture was stirred at room temperature for 2 days under argon, until CMP was consumed. The reaction mixture was concentrated by lyophilization and directly applied to a Bio-Gel P-2 column (200-400 mesh, 3 x 90 cm), and eluted with water at a flow rate of 9 mL/h at 4°C. The CMP-NeuAc fractions were pooled, applied to Dowex-1 (formate form), and eluted with an ammonium bicarbonate gradient (0.1-0.5 M). The CMP-NeuAc fractions free of the nucleotides were pooled and lyophilized. Excess ammonium bicarbonate was removed by addition of Dowex 50W-X8 (H+ form) to the stirred solution of the residual powder until pH 7.5. The resin was filtered off and the filtrate was lyophilized to yield the ammonium salt of CMP-NeuAc 17 (1.28 g, 88%). 

---