Sequential saturation analysis

Sequential saturation analysis tion reaction can be formulated as  

However, the Ab-H complex is not stable ( Section 8.4), and a high-afFmity of the antibody for H in the first step is desirable. Otherwise, L replaces H in the Ab-H eomplexes and the results resemble those of equilibrium techniques. 

Theoretically an infinite incubation period would be needed to reach perfect equilibrium in the first step (incubation with H). However, in practice, conditions are chosen so as to shorten this period to reach near-perfect equilibrium. The association and dissociation constants (fca., and /c,) are usually not known, but according to the law of mass action the higher the Ab and H concentrations, the faster Ab-H is formed. The concentration of H, however, should not exceed that of Ab. In the commercial kits (Section 14.3), the concentrations are sometimes so high that the few seconds between 

there are two processes (i) additional binding of L or H to Ab and, (ii) exchange of H by L or free H. If the incubation period of the second step is longer than needed to obtain quasiequilibrium, total [Ab-L] -I- [Ab-H] will not change, but the ratio [Ab-L]/[Ab-H] increases. On the other hand, termination of the second incubation period before near-equilibrium is reached may markedly increase the experimental error. A computer-based analysis of a series of differential equations has been published by Rodbard et al. (1971). 

Interrelationships of commonly used mathematical methods to describe AM-type immunoassays To detect the presence 

---

Fig. 3. Affinity analysis of wild-type and mutant K49VlcT of MAb HyHEHO sequentially saturated with duck lysozyme in the particle affinity assay. The abscissa (R ) is the corrected proportion of antibody binding sites filled with soluble lysozyme, and the ordinate is equivalent to [Ab] times B/F, as described in the text (Reproduced from Lavoie et al.26)...

A Zettner, PE Duly. Principles of competitive binding assays (saturation analysis) II. Sequential saturation. Chn Chem 20 5, 1974. 

This approach uses a kinetic sequential principle to carry out multicomponent CL-based determinations. In fact, when the half-lives of the CL reactions involved in the determination of the analytes in mixture are appreciably different, the CL intensity-versus-time curve exhibits two peaks that are separate in time (in the case of a binary mixture) this allows both analytes to be directly determined from their corresponding calibration plots. In general, commercially available chemiluminometers have been used in these determinations, so the CL reaction was initially started by addition of one or two reaction ingredients. Thus, in the analysis of binary mixtures of cysteine and gluthatione, appropriate time-resolved response curves were obtained provided that equal volumes of peroxidase and luminol were mixed and saturated with oxygen and that copper(H) and aminothiol solutions were simultaneously injected [62, 63],... 

The following procedure describes the preparation and analysis of the (R)-a-methylbenzylamide of (R)-a-methylbenzenepropanoic add. A flame-dried, 10-mL, round-bottomed flask equipped with a Teflon-coated magnetic stirring bar and a rubber septum is charged with 25 mg (0.15 mmol) of (R)-a-methylbenzenepropanoic acid, 31 mg (0.23 mmol) of 1-hydroxybenzotriazole hydrate, 44 mg (0.23 mmol) of 1-(3-dimethylamino)propyl-3-ethylcarbodiimide hydrochloride, and 0.50 mL of anhydrous N,N-dimethylformamide. This mixture is stirred at 23°C for 10 min, then cooled to 0°C in an ice-water bath. To the cooled solution, 24 pL (0.19 mmol) of R-(+)-a-methylbenzylamine and 86 pL (0.62 mmol) of triethylamine are added. Within 1 min, a fine white precipitate appears. The mixture is stirred for 1 hr at 0°C, then warmed to 23°C. After stirring for 20 hr at 23°C, the mixture is transferred to a 30-mL separatory funnel with 10 mL of dichloromethane. The product solution is extracted, sequentially, with four 10-mL portions of 1 N aqueous hydrochloric acid solution, 10 mL of saturated... 

Once introduced into the column, FAMEs with different carbon chain length and saturation levels move through the column at different rates and elute from the end of the column sequentially. Fatty acid standards (Nu Chek Prep or Matreya) are required to obtain the retention time for individual fatty acids in GC analysis, so that fatty acids in samples can be identified by comparing their retention times with those of the standards. In a GC run of multiple samples, standards should be analyzed prior to, during, and at the end of the sample analysis to compensate for shifts in retention times. 

Reaction Blank of 0 NH2 and Peroxidase. Twenty five mg peroxidase, 100 pi of 0 NH2, and 3 ml of H2O2 were added sequentially to 100 ml of pH 6 phosphate buffer, and stirred for 6.5 hours. The reaction was then quenched by acidifying to pH 2 with acetic acid. The colored reaction products were sorbed onto a C18 solid phase extraction cartridge, rinsed with distilled deionized water, and then eluted with methanol. The methanol was removed using rotary evaporation and the reaction products redissolved in 2 ml of DMSO-d for NMR analysis. Reaction of Humic Acid with 0 nH2 in Presence of Bimessite. Four hundred mg of the soil humic acid was added to 200 ml of H2O and adjusted to pH 6 with IN NaOH. Two hundred pi of 0 NH2 and 50 mg of bimessite were added and the reaction mixture stirred for 6 days. The reaction solution was then dialyzed, filtered through a sintered glass funnel, H" - saturated on the cation exchange resin,... 

An FIA system is especially useful in the analysis of solutions containing high levels of solids such as saturated salt solutions or dissolved fusion mixtures. The burner slot or nebulizer will not become blocked since the system is continuously and thoroughly rinsed with the carrier stream after each sample measurement. The FIA system requires less than 400 /zl of sample solution, which is much less than with continuous aspiration. Thus, FIA-FAAS is the preferred technique when only small sample amounts are available. Low sample consumption is also beneficial for routine analysis, since with fully automated sequential multi-element analysis more determinations can be performed with a given sample volume. 

The data presented here is for the reaction between MAH (40 mM) with BQCN" " (0.5 mM) in acetonitrile half-saturated with air (Table 1.12). The latter was achieved by mixing the MAH solution from which air was removed with a BQCN" solution in acetonitrile saturated with air in the mixing chamber of the stopped-flow spectrometer. The 24-point sequential analysis in this case is distinctively different from any previously presented one. In Table 1.13 we see that feapp fi> st increases to a maximum value (0.000,866s ) before decreasing slowly to 0.000,831 s at segment 8 and then increases smoothly for the remainder of the time period analyzed with a value of 0.001,356 s at segment 24. 

Fig. 14 shows a comparison of the eariy time evolution of hole injection efficiency from evaporated Au top contacts (staged evaporation) into two specimens of 40 wt% TPD/polycarbonate. Note that sequentially evaporated Au contacts were employed in order to minimize the effect of interfacial damage. Fig. 14 shows a comparison of the temporal evolution of the injection efficiency from Au into a MDP specimen as prepared (curve a) with a separate specimen of the same thickness that was vapor doped for 30 minutes in a saturated atmosphere of methylene chloride vapor just prior to analysis (curve b). Fig. 14 dearly shows that prior vapor doping increases the initially observed injection dfidency Jai/ tfsclc ns... 

---