Ninhydrin assay

Anchored amine materials can be prepared through a number of synthetic methodologies. Because of the potential importance of these materials to organic synthesis, a ninhydrin assay was developed as a rapid laboratory determination of available surface amines. The assay agreed well with expected values for aminopropyltriethoxysilane grafted onto commercial silica. The assay also distinguished between reactive amines and protonated or poisoned surface amines on co-condensed SBA-15 materials. 

Ninhydrin Assays. Ninhydrin tests were performed using a modified procedme of Taylor et al. " APS Silica (10-75 mg) of various loadings (0.857, 0.571, and 0.343 mmol NH2/g Silica) was added to phosphate buffer (5 mL, 100 mM, pH 6.5), and 1 mL of a 5% w/v solution of ninhydrin in ethanol was added to the sluny. After stirring for an hour in a boiling water bath, the mixture was allowed to cool slowly to room temperature. The silica was then filtered and washed three times with 70°C distilled water. The filtrate was collected, added to a volumetric flask, diluted to 100 mL, and the absorbance of this solution at 565 mu was measured using a UV-visible spectrophotometer. The reference solution was prepared as above with unmodified amine-free silica. Calibration standards were prepared with aliquots of a 1 mg/mL solution of APS in ethanol. 

Table 38.1. Ninhydrin assays for surface amines using grafted silicas. Determined from grafting synthesis and elemental analysis. "Typical standard deviations were...

Table 38.2. Ninhydrin assays for surface amines on APS-SBA-15 materials, all materials were alkylated after polymer removal determined from elemental analysis frefluxed in 10% HCl in ethanol for 24 hours stirred with 25 wt%...

The ninhydrin assay clearly shows that only a fraction of the total amines react with ninhydrin, suggesting that most of the amines are either protonated (and therefore... 

Pregnalatto and Sabino200 incorporated glycerine into the ninhydrin solution and reported an improved sensitivity and reproducibility enabling neomycin concentrations as low as 4 yg/ml to be determined. An automated ninhydrin assay has been described by Kaptionak, Biernacka and Pazdera O based on a modified Moore and Stein methodl l. 

Ninhydrin assay. Our method to determine reactivity towards ninhydrin was a modification of a method described previously (Moore and Stein, 1954 Moore, 1968). Briefly, dried samples were dissolved in 0.10 ml 0.1 M acetic acid and mixed with an equal volume of ninhydrin reagent (Sigma). After 15 minutes in a boiling water bath, samples were diluted with 0.80 ml ethanol/water (1 1 by vol.) and measured for absorbance at 550 nm on a flow-through spectrophotometer (Vitatron). Standards containing 0-0.6 mM leucine in 0.1 M HAc were included. Values were therefore calculated as leucine-equivalents. 

For calculation, it was assumed that 1 mol of hydroxylysinonor-leucine, dihydroxylysinonorleucine, and carboxymefhyllysine corresponded with 2, 2, and 1 mol of leucine equivalents, respectively, in the standard solutions as determined by the manual ninhydrin assay. 

The fractions were concentrated and subjected to the ninhydrin assay. Thereafter, fractions eluted with BuOH/HAc/Fl20 were combined and concentrated, as were the fractions eluted with water. The presence of cross-links was verified by TLC. 

Leone, M.M., Nankervis, R., Smith, A., and Ilium, L. (2004). Use of the ninhydrin assay to measure the release of chitosan from oral delivery systems. Int. J. Pharmaceut., 271, 241-243. 

The microwave protocol increased the reaction rate at least two- to threefold, as conversion was only 60-80% within 6 min under conventional heating. This improved coupling efficiency was duplicated with numerous amino acid derivatives and a further two peptide fragments were coupled with the Gly-Wang resin. These couplings were completed within 2 min as determined by quantitative ninhydrin assay. 

Mulholland F, Movahedi S, Haque GR, Kasumi T (1993) Monitoring tripeptidase activity using capillary electrophoresis. Comparison with the ninhydrin assay. J Chromatogr 636 63-68. 

Monitor the coupling steps by ninhydrin assay (26) or Bromophenol blue assay (27) in the case of proline to assess the presence of free amines. 

The blocking of free amino groups by acetylation was verified by the ninhydrin assay (28). 

More recent studies by Schmir and Cohen (1961) have led to a better understanding of the details and to improved yields in the tyrosine cleavage Phloretylglycine was oxidized with three equivalents of NBS in various buffer mixtures and the extent of cleavage determined both by ninhydrin assay for glycine and by the intensity of the dienone peak at 260 m/u in the ultraviolet. From the results (Table VIII) it is evident that cleavage is favored by increasing the acidity of the reaction mixture. 

After removal of excess of alkylating agent by ether extraction the reaction mixture was heated for 1 hr at 100°C. The liberated amino acid was determined by ninhydrin assay (Moore and Stein, 1948). 

Determined by ninhydrin assay for the cleaved amino acid. 

Ninhydrin Assay for Adsorbed Proteins. Measurements were made by a colorimetric procedure based on the reaction of ninhydrin with amino acids (25). The films were hydrolyzed in 5 ml of 2.5N NaOH for 2 hrs in capped plastic tubes in a boiling water bath. Then 1.5 ml of glacial acetic acid was added and mixed next I ml of ninhydrin reagent was added and mixed. [The reagent was three times more concentrated in ninhydrin, SnCb, and citrate than prescribed by Moore and Stein (25)]. The tubes were capped and boiled 20 mins more. The solution was clarified by centrifugation, and the absorbance read immediately at 570 nm on a Beckman DB spectrophotometer. If necessary, the sample was diluted with 50-50 2-propanol-water. Calibration curves (absorbance vs. fig of protein) were constructed in the 0-30 and 0-100 fig range with known amounts of each type of protein subjected to this same analysis procedure. 

All adsorption experiments were done in 0.5 mg/ml 7-globulin solutions at room temperature for at least 40 hrs, followed by at least 6 hrs of rinsing in the equilibration solvent. Adsorbed protein was determined using the ninhydrin assay. 

The listed amount of methacrylic acid was contained in the HEM A monomer used to form the poly (HEM A)/Silastic hydrogels, which had 20% grafted poly(HEMA). These hydrogels were equilibrated for 45 hrs at 37°C in 0.5mg/ml protein solutions in the listed solvents and then rinsed in the equilibration solvent, using decantation and dilution and 15 min of stirring. Ninhydrin assays were then used to determine absorbed protein. 

The three most common assays for total protein2 are the Lowry (enhanced copper), Smith (bicinchoninic acid, BCA), and Bradford (Coomassie Blue) methods. All are colorimetric methods, and are based on the generation of absorbing species in proportion to the quantity of protein present in the sample. The ninhydrin assay is a recently reported promising method. 

In a new approach for the direct determination of free and bound sialic acid, an acidic ninhydrin assay has been proposed [263]. Heating of solutions of sialic-acid-containing material with ninhydrin/acetic acid/37% HCl at 100 C yields a stable chromophore, the absorbance of which can be measured at 470 nm. 

FIG. 18. Concentration of total free amino acids (Cd-ninhydrin assay) in Cheddar cheese as a function of age (from O Shea, 1993). 

FIG. 19. Relation between the concentration of total free amino acids by the Cd-ninhydrin assay, A507, and water-soluble nitrogen for some cheese varieties (E. Olthoff, R. Schmidt, and P. F. Fox, unpublished). 

Initial cone, of amino acids in the source phase were 0.03 M carrier was 2 x 10" Af stirring rate is 500 rpm, Analysis by ninhydrin assay. 

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