Separating Amino Acids

Each amino acid is characterized by an isoelectric point , the pH at which it exists in neutral form. Differences in isoelectric points may be exploited to separate amino acids in what is termed an electrophoresis experiment. 

In the ion-exchange technique, separated amino acids exiting (eluting) from the end of the chromatography column mix with a solution of ninhydrin and undergo a rapid reaction that produces an intense purple color. The color is detected by a spectrometer, and a plot of elution time versus spectrometer absorbance is obtained. 

Post-column reaction is a common feature of many special types of analyses, the most well-known being the amino acid analyzer that uses ninhydrin with a post-column reactor to detect the separated amino acids. In general, derivatization and post-column reactor systems are techniques of last resort. In some applications they are unavoidable, but if possible, every effort should made to find a suitable detector for the actual sample materials before resorting to derivatization procedures. 

Depicted in Fig. 2, microemulsion-based liquid liquid extraction (LLE) of biomolecules consists of the contacting of a biomolecule-containing aqueous solution with a surfactant-containing lipophilic phase. Upon contact, some of the water and biomolecules will transfer to the organic phase, depending on the phase equilibrium position, resulting in a biphasic Winsor II system (w/o-ME phase in equilibrium with an excess aqueous phase). Besides serving as a means to solubilize biomolecules in w/o-MEs, LLE has been frequently used to isolate and separate amino acids, peptides and proteins [4, and references therein]. In addition, LLE has recently been employed to isolate vitamins, antibiotics, and nucleotides [6,19,40,77-79]. Industrially relevant applications of LLE are listed in Table 2 [14,15,20,80-90]. 

Miniaturized columns have provided a decisive advantage in speed. Uracil, phenol, and benzyl alcohol were separated in 20 seconds by CEC in an 18 mm column with a propyl reversed phase.29 A19 cm electrophoretic channel was etched into a glass wafer, filled with a y-cyclodextrin buffer, and used to resolve chiral amino acids from a meteorite in 4 minutes.30 A 6 cm channel equipped with a syringe pump to automate sample derivatization was used to separate amino acids modified with fluorescein isothiocyanate.31 Nanovials have been used to perform tryptic digests on the 15 nL scale for subsequent separation on capillary Electrophoresis.32 A microcolumn has also been used to generate fractions representing time-points of digestion from a 40 pL sample.33 A disposable nanoelectrospray emitter has been... 

Many times an analyte must be derivatized to improve detection. When this derivatization takes place is incredibly important, especially in regards to chiral separations. Papers cited in this chapter employ both precolumn and postcolumn derivatization. Since postcolumn derivatization takes place after the enantiomeric separation it does not change the way the analyte separates on the chiral stationary phase. This prevents the need for development of a new chiral separation method for the derivatized analyte. A chiral analyte that has been derivatized before the enantiomeric separation may not interact with the chiral stationary phase in the same manner as the underivatized analyte. This change in interactions can cause a decrease or increase in the enantioselectivity. A decrease in enantioselectivity can result when precolumn derivatization modifies the same functional groups that contribute to enantioselectivity. For example, chiral crown ethers can no longer separate amino acids that have a derivatized amine group because the protonated primary amine is... 

The information contained in the DNA (i.e., the order of the nucleotides) is first transcribed into RNA. The messenger RNA thus formed interacts with the amino-acid-charged tRNA molecules at specific cell organelles, the ribosomes. The loading of the tRNA with the necessary amino acids is carried out with the help of aminoacyl-tRNA synthetases (see Sect. 5.3.2). Each separate amino acid has its own tRNA species, i.e., there must be at least 20 different tRNA molecules in the cells. The tRNAs contain a nucleotide triplet (the anticodon), which interacts with the codon of the mRNA in a Watson-Crick manner. It is clear from the genetic code that the different amino acids have different numbers of codons thus, serine, leucine and arginine each have 6 codewords, while methionine and tryptophan are defined by only one single nucleotide triplet. 

Amino acid analysers based on ion exchange resins are available commercially. These achieve good separations of amino acid mixtures. Fluorescent derivatives of separated amino acids constitute a very sensitive means of detecting these compounds in seawater [256,258]. Fluorescent derivatives that have been studied include o-phthalaldehyde [259], dansyl [260], fluo-rescamine [261], and ninhydrin [261]. 

This method is very useful for separating amino acids found in food samples. The most effective matrix for separation is an absorbent cellulose-based filter paper. A very effective mobile phase is 70% isopropyl alcohol in water. Although the 20 amino acids are chemically very similar, they may be successfully separated by this method. Amino acids interact with the stationary phase to different extents, thus moving at different speeds. Chemical differences among amino acids that determine migration speed include molecular weight, charge, and polarity. 

Quantification of the separated amino acids is usually performed by using external calibration or the internal standard method. Due to the large differences in chemical structure exhibited by the various amino acids, there is not a single ideal standard for the overall amino acid profile. Nevertheless, a suitable internal standard must be stable to hydrolysis and offer chromatographic resolution. The most popular choices comprise norleucine, norvaline, and a-amino-n-butanoic acid (AABA) [196]. 

To separate amino acids as diastereomers two main approaches can be followed ... 

El 1. Amino acid analyzers are instruments that automatically separate amino acids by cation-exchange chromatography. Predict the order of elution (first to last) for each of the following sets of amino acids at pH = 4. 

Gonzalez de Llano et al. (47) separated amino acids from low-molecular-weight peptides by means of size-exclusion chromatography on Sephadex G-10, with water as the solvent, as a preparatory step before RP-HPLC analysis of peptides from blue cheeses soluble in 5% PTA (Fig. 1). This technique has also been used (51a) to eliminate the amino acids from the ethanol-... 

Separation of diastereoisomeric peptides by HPLC is more common. Since each diastereo-isomer has different physicochemical and biological properties, this is of great interest. Separations of diastereoiosomeric di- and tripeptides have usually been performed on reversed-phase columns. Cahill et al. (119) separated diastereoisomeric amino acids and derivatized dipeptides using esters of the /V-hydroxysuccinamide of f-butyl carbonyl-L-amino acid on Cl8 and C8 columns. Linder et al. (120) separated amino acid and peptide derivatives on an RP-C8 column, adding a metal chelate. Mixtures of DL and LD-dipeptides can be separated by RP-HPLC into two peaks, one containing LL- and DD-isomers, the other containing LD and DL-isomers. Sep-... 

Quantitative determination of the separated amino acids is achieved by their reaction with ninhydrin to produce... 

Reaction of ninhydrin with an amino acid yields a colored complex. The ninhydrin reaction permits qualitative location of amino acids in chromatography and quantitative assay of separated amino acids. 

Electrophoresis can separate amino acids by subjecting them to an electric field. The electric field applies a force whose strength and direction is dependent upon the net chaise of die amino acid. If a solution of amino acids at a pH of f underwent electrophoresis, which of the following would most likely move the furthest toward the anode ... 

Since Pasteur separated crystalline sodium ammonium tartrate manually in 1848, many researchers have worked on the subject of enantiomeric separation. In 1939 Henderson and Rule fully separated derivatives of camphor by column chromatography using lactose as a stationary phase material [1]. Gil-Av et al. [2] were able to separate amino acid derivatives on a polysiloxane-based stationary phase by gas chromatography (GC) in 1966. Since then many approaches for a successful distinction between enantiomers have been developed for capillary GC and liquid chromatography [3]. It is still a current topic for researchers searching for chiral separation with SciFinder [4] results in 812 hits and searching for chiral recognition leads to 285 hits for the year 2003 only. 

Finally, we have encountered situations where carbon-14 from a labeled pesticide has been incorporated into normal natural products including amino acids. We were interested in seeing if we could separate the normal amino acids from a protein hydrolysate on a single column with sufficient resolution to identify separate amino acids. Figure 11 shows a radiochromatogram of the separation of sixteen standard C-amino acids. Were this a product of a metabolism study, we could then isolate any of these fractions, convert the residual amino acid to the N-trifluoroacetyl 0-butyl derivative for gas chromatography ar.d further confirmation of structure. 

Enzymes such as protease in conjunction with pancreatin and amylase have been extensively used to liberate Se species from proteins for analysis [43, 57, 128, 133-136]. Relatively long times ( 24 h) are required to fully hydrolyze proteins using enzymes. However, not all Se is released as simple amino acids. Some peptides, and small molecular weight proteins remain. Thus, ultrafiltration (< 1 kDa) before analysis will be needed to separate amino acids from other material with higher molecular weight. In the presence of cysteine, selenomethionine and selenocysteine are stable to enzyme attack (Fig. 20.2). However, although large amounts of Se are released from marine tissues (30-60 percent), little (less than 10-20 percent) is characterizable by HPLC-ICP-MS. 

There are approximately 20 separate amino acids that form various proteins of immense chain length, some with as many as 2000 units of different combinations in their chains. Each protein has its own characteristic shape some are long chains, some are spirals and some are more ball-like. Their shape often determines the specific and characteristic properties of the particular protein. 

Protein chain attacked by the water Separate amino acids... 

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