Ninhydrin Kaiser Test

Comments Commercially available. Detects primary amines to a limit of 5 pmol/g. Provides an intense blue color or in some cases a brown/reddish brown color. First used to detect incomplete coupling reactions in peptide synthesis. 

Comments Commercially available. A noninvasive qualitative and quantitative test. Provides a deep blue color. 

Other Reagents. Acetaldehyde (for primary and secondary amines) and acetone (for secondary amines).  

Comments. Commercially available. Detects primary and secondary aliphatic amines as well as primary aromatic aminesto a limit of 5 pmol/g for aromatic amines. Provides a green/blue color ° or a red color. 

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Fmoc-Rink-Nle-pMeBHA-resin (1 g, 0.55 mmol g ) was swollen for 1.5 h in NMP in a reaction vessel equipped with a sintered glass bottom, and placed on a shaker. The Fmoc group was removed with 20% piperidine in NMP (8mL, 2 x 15 min) and after washing with NMP (8mL, 5 x 2 min), the Fmoc removal was monitored by the ninhydrin Kaiser test. Coupling of building blocks such as iV -Fmoc-/Va>-carb-oxyalkyl(OAl)Xaa-OH or /V -Fmoc-/V" -aminoalkyl(Aloc)Xaa-OH, or N -alkylated amino acids such as... 

The Kaiser test uses three solutions ninhydrin (5 g in 100 ml of ethanol), phenol (80 g crystalline phenol in 20 ml ethanol), and pyridine/KCN [dilute 2 ml of 0.001 mol/liter aqueous solution of KCN (potassium cyanide) with 98 ml of pyridine]. The test is performed by transferring a few resin beads to a 6 x 50-mm glass test tube. The beads are washed with ethanol and a drop of each of the three testing solutions is added. The tube is then heated to 100° for 5 min in a heating block. Blue (brown for proline)-colored beads and solution (positive test) indicate the presence of free amino groups on the resin. Colorless beads (negative test) indicate complete coupling. The quality of phenol is important. Impure phenol may result in a false-positive Kaiser test. 

In the commonly used Kaiser test, a few milligrams of resin beads is withdrawn from the reaction, thoroughly washed with a range of solvents, and treated with stock ethanolic solutions of ninhydrin and phenol followed by a solution of KCN in pyridine at 100 °C for 10 min. If the beads turn deep blue in color, there are free primary amine... 

Kaiser test solutions (ninhydrin, pyridine, phenol) (see Note 1). 

Kaiser test. Solution A 5% Ninhydrin in ethanol (w/v). Solution B 80% phenol in ethanol (w/v). Solution C KCN in pyridine (2 mL 0.001 M KCN in 98 mL pyridine). After washings, sample a few resin heads in a small glass mbe and add 2 drops of each of the above solutions. Heat to 120°C for 4-6 min. Blue resin beads indicate the presence of resin-bound free amines. 

The monitoring of solid-phase synthesis in a real time quantitative manner has always been a goal since invention of the process. Many qualitative color tests exist, principal amongst these being the ninhydrin-based Kaiser test.f 1 The Kaiser test is less sensitive when the N-terminal is a secondary amine and for this reason many other tests have been applied.t °l Although a quantitative Kaiser test has been proposed,t l it has never achieved prominence as a routinely applicable method and the results are not usually available in time to influence progress of the reaction. 

The resin gave a positive Kaiser test (ninhydrin). 

The resin gave a negative Kaiser test (ninhydrin). The supported product (431) can then be processed further to afford the bicycle (432) [351]. 

The qualitative ninhydrin, or Kaiser, test can be used for monitoring functionalization of an amino-resin by a linker. Alternatively, the quantitative ninhydrin test can be used to determine the loading of an amino resin. 

Kaiser test, ninhydrin test, a simple and most frequently used method of on-resin monitoring in SPPS. A positive color reaction, performed with a small aliquot of the resin material, indicates unconverted amino groups. Samples containing <0.5% of unreacted amino groups can usually detected within minutes. A modified version of the Kaiser test allows quantitative analysis [E. Kaiser et al.. Anal. Biochem. 1970, 34, 595 V. K. Sarin et al.. Anal. Biochem. 1981, 117,147]. 

The Kaiser test is an adaption of the ninhydrin test to sohd phase conditions and allows the visual detection of free amines in resin-bound substrates. The possibility of qualitatively or quantitatively tracking the reaction progress of polyamine modification makes it a very helpful tool for sohd phase chemistry. The general solution phase methods (e.g., thin layer chromatography) are not suitable. Here, the Kaiser test was used to additionally... 

Kaiser test ninhydrin, phenol, ethanol, pyridine, potassium cyanide (KCN). 

Kaiser test is a rapid, qualitative ninhydrin-based test for detection of free primary amines. Put a few beads of your resin in a 1.5 mL tube and add 1-2 drops of Kaiser test solutions 1-111. Incubate 5 min at 95 °C. Blue solution indicates the presence of free amines. Notice The test does not work in case of proline, because of its nature as secondary amine ... 

After the reaction times given in Scheme 38, the respective couplings were quantitative according to chloranil and the Kaiser ninhydrin test and/or HPLC of small cleavage. The peak area of the crude Fmoc-deprotected dodecapeptide was 90% (HPLC k=214nm). The pure cyclic peptide was obtained in an overall yield of 31%. Cyclosporin O was synthesized by a similar procedure.129 ... 

Qualitative color tests are frequently used to follow reactions to assure completion. The Kaiser ninhydrin test is the best known of these [15], An improved method for the detection of secondary amines has been reported [16], The Fmoc method for assuring complete deprotection of an amine is in wide use [13],... 

Linker attachment An amino-functionalized polystyrene or PEG-polystyrene resin may be used as starting material. First, 5-(4-formyl-3,5-dimethoxyphe-noxy)valeric acid (PALdehyde) [122] (4equiv.) and HATU [123] (4equiv.) were dissolved in DMF, DIEA (8 equiv.) was then added, and, after 1 min of pre-activation, this solution was added to the resin (1 equiv.). Coupling was allowed to proceed at 25 °C for 2 h, after which the Kaiser ninhydrin test [81] was negative. 

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