Dansyl amino acid standards

Figure 5. Chromatogram of dansyl amino acid standards. Column fused-silica capillary (0.32 mm i.d., 1.3 m length) packed with Aquapore RP-300 (10 jim) Mobile phase linear gradient from 5% to 32% 2-propanol in 0.05 M formic acid/0.06 M acetic acid buffer (pH 3.2) in 200 min, 2.0 pL/min, 55 C Sample injection 130 nL, 50-100 pmol per component.

Period 2 Part B—Work up the dansyl hydrolysate and spot on the TLC plate with standard dansyl amino acids. Part A. 1—Work up peptide hydrolysate and prepare FMOC derivatives of amino acids for analysis by HPLC or CE. Part A.2-If applicable, develop paper chromatogram in solvent system. 

Solvent 3. Ethyl acetate-methanol-acetic acid, 2 1 1 (v/v/v) Standard mixture of dansyl amino acids in acetone, 1 mg/mL each Phe, Leu, Gly, Ala, Val. 

Draw a picture in your notebook of the polyamide thin-layer plate exposed under UV light after each of the two or three solvent developments. These pictures should look similar to Figure E2.7. Three fluorescent areas should be evident after solvent 2 however, better separation is achieved by solvent 3. A blue fluorescent area at the bottom of the plate is dansic acid (DNS-OH), which is a hydrolysis product of dansyl chloride. A blue-green fluorescent spot about one-third to one-half up the plate is dansyl amide (DNS-NH2), which is produced by reaction of dansyl chloride with ammonia. A third spot, which usually fluoresces green, is the dansyl derivative of the NH2-terminus amino acid. Note the positions of the standard dansyl amino acids and compare with the unknown. What is the identity of the NH2-terminal amino acid Are any other fluorescent spots evident on the plate Using polarity or nonpolarity, try to explain the position of each molecule on the thin-layer plate. 

The earliest applications of acoustic levitation in analytical chemistry were concerned with the development of various steps of the analytical process. Thus, Welter and Neidhart [72] studied the preconcentration of n-hexanol in methanol by solvent evaporation and the liquid-liquid extraction of n-hexanol from water to toluene in a levitated droplet, which they found to be efficient when using GC-FID with n-pentanol as internal standard. Solvent exchange of fluorescein from methylisobutyl ketone to aqueous sodium hydroxide was also accomplished. Sample concentration in an acoustically levitated droplet prior to injection into a CE equipped with an LIF or UV detector has also been accomplished [73,118]. The target analytes (namely, dansylated amino acids) were concentrated in the levitated drop and a limit of detection of 15 nM — much lower than the 2.5 pM achieved by hydrodynamic injection without preconcentration — was achieved following CE separation and quantification. For this purpose, 36000 sample droplets 2.3 pi in volume each were sequentially positioned in the acoustic Ievitator and evaporated. This example illustrates the potential of acoustic levitation for coupling to any type of detector for micro- or nanotrace analyses. 

Amino acid sequencing was performed (304) by initial Raney nickel treatment of cortinarin A followed by partial hydrolysis and degradation of the resulting linear peptide employing dansylation of the terminal amino acid. Dansyl amino acids were identified by comparison with standards using two-dimensional TLC on polyamide plates. The amino acid sequence was also determined by the mass spectrometry method (Scheme 61). The sequence reported (330) in 1986 appeared to be a reversal of that shown previously (304). 

However, although it is possible to detect very low concentrations of dansyl amino acids fluorimetrically, use of the dansyl reaction for the quantitative analysis of amino acids has proven difficult in the past. Many factors influence the yield of the dansylation reaction In addition, side reactions are known to occur. During dansylation, a second molecule of dansyl chloride may combine with the acidic group of the ammo acid and the product then readily hydrolyzes in the alkaline conditions of the reaction medium (Boulton, 1968) This situation is potentially even more complex when mixtures of ammo acids are to be derivatized. In such cases, the yield of the dansylation reaction for each ammo acid constituent of the mixture must be estimated. This has required, in the past, time-consuming parallel experiments involving dansylation of standard ammo acid mixtures... 

FABMS has been employed to identify and quantify dansyl amino acids (8) formed in a procedure for the N-terminal analysis of proteins (57). This sensitive method involves the reaction of the protein with dansyl chloride, followed by acid hydrolysis. Previously the characteristic involatile N-terminal amino acid derivative (8) was analyzed either by TLC or high-voltage electrophoresis but FAB ionization now permits rapid identification of the protonated molecular ion. The responses of this and other characteristic ions were monitored employing dansyl amino butyric acid as an internal standard, enabling the dansyl derivatives (8) to be determined quantitatively at a level of 0.1 nmol. 

Period 1 Part A—Begin acid hydrolysis of peptide. Part B—Complete dansyl chloride reaction and begin acid hydrolysis of dansyl peptide. Part A.2—if applicable, prepare paper chromatogram by applying standard amino acids. 

The amino-terminal (N-terminal) residue of a protein can be identified by reacting the protein with a compound that forms a stable covalent link with the free a-amino group, prior to hydrolysis with 6 M HC1. The labeled N-terminal amino acid can then be identified by comparison of its chromatographic properties with standard amino acid derivatives. Commonly used reagents for N-terminal analysis are fluorodinitrobenzene and dansyl chloride. If this technique was applied to the oligopeptide above, the N-terminal residue would be identified as Val, but the remainder of the sequence would still be unknown. Further reaction with dansyl chloride would not reveal the next residue in the sequence since the peptide is totally degraded in the acid hydrolysis step. 

Figure 2 illustrates the structure of the product resulting from the derivatization of an amino acid with the above-mentioned reagents. Mixtures of derivatives with the most commonly used reagents (dansyl, DNP, PTH) are readily provided in kits, to be directly used as reference standards in HPLC analysis. 

Amino acids were derlvatlzed with 1-dlmethylamlnonaphthalene-5-sulfonyl chloride (dansyl chloride) according to the procedure of Tapuhl and coworkers (48-50). A 10 M stock solution of twenty common -amlno acids (Sigma Chemical Co., St. Louis, MO) was prepared by dissolving the carefully weighed standards in 0.1 M aqueous hydrochloric acid. Aliquots of this solution were transferred to conical vials, evaporated to dryness, and redlssolved In 500 pL aqueous buffer (0.04 M lithium carbonate, pH 9.5). A 500 jL volume of dansyl chloride solution (5 x 10 3 m in acetonitrile) was added, and the derlvatlzatlon was allowed to proceed In the dark at 35"C for one hour. The reaction was terminated by the addition of 2% methylamlne hydrochloride, and the derlvatlzed sample was analyzed Immediately by mlcrocolumn liquid chromatography with UV-absorbance and LIF detection. 

The DANSYL reagent reacts with both primary and secondary amines to produce fluorescent derivatives. These derivatives are very stable when stored in the dark and authentic standards are commercially available. One of the problems with this derivatization chemistry is hydrolysis of the reagent and the formation of multiple derivatives for several of the amino acids. In addition, there is fluorescence quenching in polar solvents, so detection limits using reversed-phase chromatography with UV detection are often comparable to that obtained by fluorescence. The limit of detection using UV detection is 100 pmol. 

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