Mobility, of amino acids

The availability of gluconeogenic precursors, particularly gluco genic amino acids, significantly influences the rate of hepatic glu cose synthesis. Decreased levels of insulin favor mobilization of amino acids from muscle protein, and provide the carbon skele tons for gluconeogenesis. 

When the cell requires instructions for protein production, part of the code on DNA, starting at an initiator and ending at a stop codon, is converted into a more mobile form by transferring the DNA code into a matching RNA code on a messenger ribonucleic acid (mRNA), a process known as transcription. The decoding, or translation, of mRNA then takes place by special transfer ribonucleic acids (tRNA), which recognize individual codons as amino acids. The sequence of amino acids is assembled into a protein (see Proteins section). In summary, the codes on DNA... 

The copper complex is very stable at neutral pH, but it fades very rapidly in the presence of hydrogen ions. Other complex formers such as tartaric acid or citric acid and thiourea interfere with the reaction and, therefore, should not be included in mobile phases used for the separation of amino acids [3]. 

FIGURE 8.7 Effect of pH on retention of amino acids. Column and flow rate Same as Fig. 8.1. Mobile phase 10 mA1 potassium phosphate with SO mM HFIP pH as indicated (adjusted prior to the addition of HFIP). 

Electrostatic effects have long been recognized in commercial HPLC columns for SEC of proteins (15,21,22). The usual remedy is to add 100 mM salt to the mobile phase. This works here too the Lys and Asp peaks collapse into the Gly peak with 100 mM salt (Eig. 8.8). High concentrations of sodium sulfate were added to determine the role played in SEC by hydrophobic interactions (sodium sulfate, a structure-forming salt, strengthens such interactions). Sodium sulfate increased the retention only of the most hydrophobic amino acids to any extent, and then only when the concentration approached 1 M. Clearly, hydrophobic interaction cannot account for the elution order of amino acids on PolyHEA. 

FIGURE 8.13 SEC of casein hydrolyzates. Numbers above the peaks refer to the number of amino acid residues in the typical peptide in the indicated fraction. Column PolyHEA, 200 X 9.4 mm 5 /zm, 200 A. Flow rate 0.5 ml/min. Mobile phase 50 mtA Formic acid. Detection A250. Samples (A) Pancreatin hydrolyzate and (B) tryptic hydrolyzate. (Adapted from Ref. 29 with permission from Silvestre et of. Copyright 1994, American Chemical Society.)... 

Electrophoresis is used primarily to analyze mixtures of peptides and proteins, rather than individual amino acids, but analogous principles apply. Because they incorporate different numbers of amino acids and because their side chains are different, two peptides will have slightly different acid-base properties and slightly different net charges at a particular pH. Thus, their mobilities in an electric field will be different, and electrophoresis can be used to separate them. The medium used to separate peptides and proteins is typically a polyacrylamide gel, leading to the term gel electrophoresis for this technique. 

The great leap forward for chromatography was the seminal work of Martin and Synge (7) who in 1941 replaced countercurrent liquid-liquid extraction by partition chromatography for the analysis of amino acids from wool. Martin also realized that the mobile phase could be a gas rather than a liquid, and with James first developed (8) gas chromatography (GC) in 1951, following the gas-phase adsorption-chromatographic separations of Phillips (9). 

Fig. 7-9. Separation of amino acids after derivatization 5 with OPA and mercaptoethanol. Column Superspher 100 RP-18 (4 pm) LiChroCART 250-4, mobile phase 50 mM sodium acetate buffer pH 7.0/methanol, flowrate 1.0 ml min temperature 40 °C detection fluorescence, excitation 340 nm/emission 445 nm. Sample amino acid standard sample (Merck KGaA Application note W219180).

Shlf-Test M4.1A A pair of amino acids is separated in a column in which the stationary phase is saturated with water and the mobile phase is methanol, Cl 1,011. The more polar the acid, the more strongly it is adsorbed by the... 

A review on TLC and PLC of amino adds, peptides, and proteins is presented in the works by Bhushan [24,25]. Chromatographic behavior of 24 amino acids on silica gel layers impregnated tiraryl phosphate and tri-n-butylamine in a two-component mobile phase (propanol water) of varying ratios has been studied by Sharma and coworkers [26], The effect of impregnation, mobile phase composition, and the effect of solubility on hRf of amino acids were discussed. The mechanism of migration was explained in terms of adsorption on impregnated silica gel G and the polarity of the mobile phase used. 

A simple and rapid method of separating optical isomers of amino acids on a reversed-phase plate, without using impregnated plates or a chiral mobile phase, was described by Nagata et al. [27]. Amino acids were derivatized with /-fluoro-2,4-dinitrophenyl-5-L-alanine amide (FDAA or Marfey s reagent). Each FDAA amino acid can be separated from the others by two-dimensional elution. Separation of L- and D-serine was achieved with 30% of acetonitrile solvent. The enantiomers of threonine, proline, and alanine were separated with 35% of acetonitrile solvent and those of methionine, valine, phenylalanine, and leucine with 40% of acetonitrile solvent. The spots were scraped off the plate after the... 

Figure 7 Separation of amino acids by high performance ion exchange Beckman Model 6300/7300 Amino Acid Analyzer. Column 20 cm sodium high performance column. Mobile phase Na-E, Na-F, Na-D, and Na-R regenerant, with buffer changes occurring at ABj and AB2. Flow rate 20 ml/hr. Detection Ninhydrin post-column (absorbance at 440 nm and 570 nm). Temperature 49°C rising to 79°C at AT . Note that the separation requires about 40 min. Compare to Figure 6. (Application Note A6300-AN007, reproduced with permission from Beckman Instruments Fullerton, CA.)...

Factors that can predispose patients to developing metabolic bone disease include deficiencies of phosphorus, calcium, and vitamin D vitamin D and/or aluminum toxicity amino acids and hypertonic dextrose infusions chronic metabolic acidosis corticosteroid therapy and lack of mobility.35,39 Calcium deficiency (due to decreased intake or increased urinary excretion) is one of the major causes of metabolic bone disease in patients receiving PN. Provide adequate calcium and phosphate with PN to improve bone mineralization and help to prevent metabolic bone disease. Administration of amino acids and chronic metabolic acidosis also appear to play an important role. Provide adequate amounts of acetate in PN admixtures to maintain acid-base balance. 

TLC has similar applications to paper chromatography. The stationary phase is a coating, such as silica gel, on a glass or plastic plate. Depending on the TLC plate used, components may be separated based on differences in molecular weight, charge, or polarity (see Chapter 11). TLC with a 70% isopropyl alcohol mobile phase and a silica gel plate is an effective substitute for paper chromatography separation of amino acids. Nucleotides may be separated on a special silica gel plate and a 20% ethanol (in water) mobile phase. 

HPLC is frequently employed in the analysis of amino acids, peptides, proteins, nucleic acids, and nucleotides. HPLC is also often used to analyze for drugs in biological samples (see Workplace Scene 16.2). Due to the complex nature of the molecules to be analyzed, these techniques tend to be more complex than HPLC applications in other areas of analytical chemistry. For example, separation of nucleotides or amino acids is more difficult than testing for caffeine in beverages, even though the same instrument and same general methods would be employed. A variety of columns and mobile phases are regularly employed. 

Cellulose is itself polar in nature and can cause some adsorption, which may result in the tailing of zones. However, this adsorptive effect may contribute to the separation process in some instances and the use of a polar mobile phase can enhance this effect further, e.g. the separation of amino acids using an aqueous solution of ammonia as the mobile phase. The combination of partition and adsorption generally influences separations on cellulose thin-layer plates, which have superseded paper chromatography in most instances and offer increased speed and resolution. 

Two-dimensional separations permit the resolution of large numbers of amino acids present in a sample and those having a similar mobility in one dimension will usually be separated from each other in the second. This is especially useful in the detection of components that are present only in low concentrations and might be obscured in one dimension by other amino acids that are present in higher concentrations. 

Applicability of CZE to the Edman phenylthiohydantion derivatives of amino acids (140) is limited because the neutral amino acids cannot be resolved by this method and by the reduced thickness of the sample requiring relatively high concentrations of the fluorescent material for detection. These limitations may be overcome by a micellar technique that confers mobility to neutral 140 species and by application of thermotropic detection that allows one to detect a few tens of fmol of the derivative, obtained after injecting ca 0.5 nL, at a concentration of ca 1 p-M330. 

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