Buffers, ACES,

Preparation of PhAcOZ amino acids proceeds from the chloroformate, and cleavage is accomplished enzymatically with penicillin G acylase (pH 7 phosphate buffer, 25°, NaHS03, 40-88% yield). In a related approach, the 4-ace-toxy derivative is used, but in this case deprotection is achieved using the lipase, acetyl esterase, from oranges (pH 7, NaCl buffer, 45°, 57-70% yield). 

The choice of the chromatographic system depends on the chemical character of the extracts being separated. The mobile phase should accomplish all requirements for PLC determined by volatility and low viscosity, because nonvolatile components (e.g., ion association reagents and most buffers) should be avoided. It means that, for PLC of plant extracts, normal phase chromatography is much more preferable than reversed-phase systems. In the latter situation, mixtures such as methanol-ace-tonitrile-water are mostly used. If buffers and acids have to be added to either the... 

Removal of the 4-chlorobenzyl protecting group of a protected cryptand was performed under buffered condtions 210 in the presence of sodium ace-... 

Figure 27. Plots of the permeation current density vs. the square root of the Tafel reaction current density [Eq. (33)], recorded in ace-tate/acetic acid buffers at pH = 6 and 30°C, using carbon steel membranes of 1.0 and 0.5 mm thickness. ...

Both D-[l- C]xylose and D-[5- C]arabinose were exposed to a concentrated phosphate buffer solution (pH 6.7). 1-Hydroxy-2-propanone (ace-tol) was distilled from the heated solution. Radioassay indicated that similar labeling [3- C] occurred in the acetol from both pentoses, with loss of the configurational difference thus, a 3-ketopentose or its enediol was suggested as an intermediate. Further work with 3-0- and 6-0-methyl-D-glucose and with 1-0-methyl-D-fructose indicated that /3-elimination from a 3-ketose or, in the case of a hexose, from a 3-ketose or a 4-ketose, or both, tautomerization of the resulting a-diketone to a /3-diketone, and hydrolytic cleavage are essential steps in the formation of acetol. 

The buffer constituents are also important for measuring the proper electrophoretic mobility. So far, phosphate, acetate, borate, citrate, formate, HEPES, CHES, MES, CAPS, AMPSO, TAPS, Tricine, MOPS, and ACES have been used. We reported that the influence of these ingredients on the mobility of some drugs was negligible (20). Also, Gluck Cleveland (32) corrected the mobility of solutes using the mobility of toluenesulfonic acid, whose charge,... 

In one of the earlier papers, Sun et al. (48) estimated the binding constants of ibuprofen, flurbiprofen, and ketoprofen to HSA and BSA using the mobility-shift mode of ACE. In this case the drugs were actually used as additives to the background buffer to improve the separation of albumin proteins. This is the opposite approach to what is presented in the next... 

Immobilizing heparin and heparan sulfate onto fused-silica capillaries using biotin-neutravidin conjugation has been applied to ACE analysis (73). These capillaries exhibit markedly reduced electroosmotic flow and were able to distinguish heparin-binding peptides that differed only in the stereochemistry or type of amino acid residue. No resolution of these peptides was observed if heparin or heparan sulfate was included in the buffer phase. 

Fig. 6 ACE analysis of a purified heparin-binding peptide fragment of SAP. Demonstration of interaction with heparin. The peptide fragment was analyzed at 20 kV (detection at 200 nm) after a 5-s injection in the presence of the indicated concentrations of heparin in the electrophoresis buffer (0.1 M phosphate, pH 7.5). (From Ref. 71.)...

Fig. 10 ACE using an etched capillary with heparin bound, (a) SLPI concentration, 10 mg/mL. ACE condition etched capillary, 75-/rm ID X 55 cm (47 cm from injection to detection window), heparin bound via silane spacer. Injection mode gravity, height 55 cm, time 15 s. Washing and elution mode pressure injection, 2 psi, 300 s. Buffer A, 25 mM sodium phosphate, pH 7.4 buffer B, buffer and 1.0 M NaCl. Detection wavelength, 220 nm. (b) ATIII concentration, 4.5 mg/mL. (c) Bovine serum albumin, 0.3 mg/mL. (From Ref. 85.)... 

Fig. 11 ACE step elution of SLPI by various concentrations of NaCl containing buffer from heparin-bound etched capillary. NaCl concentration in elution buffer see Fig. 10 for conditions. (From Ref. 85.)...

Fig. 6 Evaluation of phosvitin binding to monoclonal anti-phosphoserine antibody by ACE using mobility-shift analysis and UV detection. Peaks M, internal peptide marker mAb, free monoclonal anti-phosphoserine antibody mAb-hpAg complex, monoclonal anti-phosphoserine antibody complexed with homopolyvalent phosvitin antigen. The buffer contained phosvitin within a concentration from 0 to 60 /xM. (Reprinted with permission from Ref. 27. Copyright 1997 Academic Press.)... 

General CE problems (e.g., wall interactions of proteins) have been discussed elsewhere. All ACE techniques working with a ligand added to CE buffer (classical ACE, Hummel-Dreyer principle, vacancy peak analysis) imply the potential problem that any continuously infused matrix can increase background noise and, even worse, deteriorate the ionization of the analyte due to competition. 

Since ACE is based mostly on the calculation of migration-time shifts of analytes upon addition of an interaction partner, migration-time reproducibility is extremely important. If the calculation is based on mobilities, this problem is certainly reduced. However, stable buffer electrolyte conditions are mandatory, which may be critical in sheath flow setups. 

Fig. 3 ACE/MS of a synthetic all-D, Fmoc-DDXX library of 100 tetrapeptides using vancomycin as the receptor. (A-D) Selected ion electropherograms for the masses indicated (E) reconstructed ion electropherograms for runs without (left) and with (right) vancomycin in the electrophoresis buffer. See text for conditions and further explanations. (Reprinted with permission from Ref. 38. Copyright 1995 American Chemical Society.)...

In Ref. 42 a similar approach was chosen as in Ref. 39 using stereoisomers of the type Fmoc-L-Asp-L-Asp-D-Xaa-D-Xaa (Xaa = Gly, Ala, Phe, His, Ser, Tyr). Interestingly, in part the findings are different. The ACE/MS hyphenation caused a number of practical problems affecting the reliability of the system. Surprisingly, the authors faced problems with positive ESI and were forced to use negative ionization. Because of the use of the nonvolatile Tris buffer, crystallization problems occurred frequently. Only high-EOF conditions prevented this knockout scenario. However, the description of problems and related solutions is very instructive. 

FIGURE 2.4 Separation of strong bases on a classical RP and on a shielded phase. Columns Prontosil CIS H Prontosil CIS ace EPS (150x4.0mm). Mobile phase MeOH/phosphate buffer 20mM pH=7, 65/35 v/v, 40°C. Analytes 1, uracil 2, protriptyline 3, nortriptyline 4, doxepine 5, imipramine 6, amitriptyline 7, trimipramine S, clomipramine. 

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