Amino acids water retention

Lejon et al., (3)a derivatives ofamino acids in octanol/water dG = RTln f f = fraction buried/accessible amino acids HPLC retention times for nine combinations of three different pH and three eluent mixtures (9 descriptors)... 

Citric acid is used in carbonated beverages to provide tartness, modify and enhance flavors, and chelate trace metals. It is often added to jams and jellies to control pH and provide tartness. It is used in cured and freeze-dried meat products to protect the amino acids (qv) and improve water retention. Bakers use it to improve the flavor of fmit fillings in baked goods. Because citric acid is a good chelator for trace metals, it is used as an antioxidant synergist in fats and oils, and as a preservative in frozen fish and shellfish (7) (see Antioxidaisits). 

For most free amino acids and small peptides, a mixture of alcohol with water is a typical mobile phase composition in the reversed-phase mode for glycopeptide CSPs. For some bifunctional amino acids and most other compounds, however, aqueous buffer is usually necessary to enhance resolution. The types of buffers dictate the retention, efficiency and - to a lesser effect - selectivity of analytes. Tri-ethylammonium acetate and ammonium nitrate are the most effective buffer systems, while sodium citrate is also effective for the separation of profens on vancomycin CSP, and ammonium acetate is the most appropriate for LC/MS applications. 

It is desirable that the equilibrium constant for a solute be not zero or very large lest there be no net retention or near infinite retention. The catch comes in the fact that liquids, which are relatively good solvents for a given type of molecule are also solvents for each other. This means the risk involved is by washing off the stationary phase with the mobile phase. Yet liquid-liquid methods offer much promise for relatively nonvolatile but soluble molecules and their separation of one from the other. The discovery of liquid-liquid chromatography earned Martin and Synge the Nobel Prize when they applied it to amino acids with water mobile phases and organic liquid stationary phases. 

Serine hydroxymethyl transferase catalyzes the decarboxylation reaction of a-amino-a-methylmalonic acid to give (J )-a-aminopropionic acid with retention of configuration [1]. The reaction of methylmalonyl-CoA catalyzed by malonyl-coenzyme A decarboxylase also proceeds with perfect retention of configuration, but the notation of the absolute configuration is reversed in accordance with the CIP-priority rule [2]. Of course, water is a good proton source and, if it comes in contact with these reactants, the product of decarboxylation should be a one-to-one mixture of the two enantiomers. Thus, the stereoselectivity of the reaction indicates that the reaction environment is highly hydro-phobic, so that no free water molecule attacks the intermediate. Even if some water molecules are present in the active site of the enzyme, they are entirely under the control of the enzyme. If this type of reaction can be realized using synthetic substrates, a new method will be developed for the preparation of optically active carboxylic acids that have a chiral center at the a-position. 

In Chapter 6, an attempt was made to identify suspect physiological cross-links, tentatively assigned from HPLC dafa, in addition to those already established in literature. Two novel cross-links, denoted chromatographic fractions IV and V-2, were purified from bovine root dentin, and the structure of V-2 was elucidated. During the analysis of human denfin as described in Chapter 5, V-2 appeared below detection level. A peak was observed for IV. Part of the material with the same retention as IV was not retained on cellulose in butanol/acetic acid/water = 4 1 1 (vol.). Co-elution of a non-cross-linking amino acid with IV could therefore not be excluded, and the results were omitted. 

Fio. 38. Plot of the algorithm of the retention factor, k, and log P, the water-/i>octanol partition coefficient of eight amino acids. The chromatographic data were obtained on 3 ftm LiChrosorb kP-8, 230 x 4.6 mm i.d. eluierit 0.1 M aqueous phosphate buffer, pH 6.7, T 70 C. Eluites Trp, tryptophan Phe, phenylalanine Leu, leudne Val. valine Tyr, tyrosine Lys, lysine Ala, alanine Gly, glycine. Reprinted with permission from Molnar and Horvith QOS). 

The opposite scheme is to employ a C]8 resin (e.g., C 8 Sep-Pak from Waters Associates) in which the eluent will contain the amino acids while lipids and large proteins will be retained on the column. This procedure has been called into question, however (22). It has been suggested that partial retention of the hydrophobic amino acids can occur. This is especially a problem if norleucine (or any other hydrophobic amino acid, e.g., norvaline) is employed as an internal standard. Its partial retention on the C18 column will skew the apparent recoveries for all the other amino acids. This points to a particular problem for amino acid analysis There is no ideal choice for an internal standard. A subsequent section of this chapter will address the issue of internal standards in more detail. 

Anon (1996b) Retention time table for amino acids using the AccQ-Tag method. Waters AccQ-Tag solutions July, 2 pages. Available as a downloadable file from the Waters website (http //www.waters.com). 

Method. A standard amino-acid analyzer (Technicon or an equivalent) may be used. The reagents for development and the buffers are prepared as for analysis of amino acids. The analytical column (24 cm X 0.57 cm) consists of Zeocarb 226-4.5% DVB (average particle diameter, 24 jum). The two buffers are prepared by dissolving 8.74 g of potassium citrate, 60.36 g of potassium chloride, 10 ml of Brij and 100 ml of n-propanol (for the first buffer, 140 ml of n-propanol for the second buffer) in enough water to make a total volume of 11. The pH of each buffer is 7.4. For analysis the sample is adjusted to pH 7.4 and an aliquot portion is applied to the column. The column temperature is maintained at 43 °C for 103 min and is automatically switched to 75 °C for the remainder of the run. The flow-rate of the buffer is 42 ml/h. The first buffer is automatically replaced by the second after 120 min. The second buffer is necessary for the separation of tryptamine and cadaverine. The use of the increased temperature results in a shorter elution time. The retention times of some basic amino acids and amines are listed in Table 4.3. Absorption is monitored at 570 nm with a 1,5-cm flow cell. 

Although the majority of reports of macrocycles in analytical chromatography have involved ligand association with the stationary phase, their use as mobile phase constituents has also been investigated. Lamb and Drake [11] showed that addition of water-soluble crown ethers to the mobile phase altered the retention of alkali metal cations on an underivatized reversed phase column. Nakagawa et al. [63-66] also used crown ether-containing mobile phases in the separation of protonated amines, amino acids and peptides, and [1-lactam antibiotics. 

Q3 Vasopressin is a small peptide hormone consisting of nine amino acids, most of which is synthesized in neurosecretory cells of the supraoptic nucleus of the hypothalamus. Small quantities are also produced in the neighbouring paraventricular nucleus. The hormone is transported down the axons of the neurosecretory cells via the infundibulum to the posterior pituitary, where it is stored until release into the blood is triggered by nerve impulses from the hypothalamus. Vasopressin is better known as antidiuretic hormone (ADH). The name vasopressin relates to its vasoconstrictor action, which increases pressure in the vascular system. This action was discovered before its effects on water retention were known. 

Polar molecules are generally more soluble in water than nonpolar molecules, and therefore solubility values can also be useful in predicting retention times. For example, some amino acids (e.g., glycine, alanine, and others containing nonpolar side chains) are not very soluble in water thus, on reversed-phase columns washed with only aqueous buffers, such compounds would be expected to interact with the nonpolar packing and be retained. Elution would be promoted by increasing the organic composition of the elution buffer. 

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