Amino acid refractivity index

Refractive-index detection is seldom used for many of the same reasons just mentioned for the spectroscopic detection of amino acids in their native forms. In fact, these problems are even more severe. Refractive-index detection has almost no selectivity whatsoever. Nearly every sample component passing the detector will register a signal. Also, this makes refractive index entirely incompatible with gradient elution. Even for isocratic separation, and detection of only a select few amino acids, refractive index can be very troublesome because of the detector s tendency to drift due to temperature changes in the laboratory (perhaps newer models have fixed this problem ). Finally, detection limits tend to be very poor for refractive-index detection. 

Often it is required to detect compounds with no or only very weak chromophores such as sugars and amino acids. Refractive index detectors and mass sensitive detectors can be used but they are relatively insensitive in the context of biological sample concentrations. Indirect detection using a UV or fluorescent eluent can also be employed. However, the most common approach is the use of derivatisation. Derivatisation of some chemically reactive moiety on the analyte can be performed in two modes. In post-column derivatisation the sample is separated first and then reacted with a flowing stream of derivatising reagent being pumped into... 

Laser-based refractive index detector, Cuprammonium reagent,4-Aminobenzoic acid reagent, Indirect detection methods for cyclodex-trins, and sugar phosphates Reversible derivatization using 2-amino-pyridine ... 

An ultraviolet-laser based thermo-optical absorbance detector for micrometer capillaries was used by Qi et al. [76] to monitor the separation of a mixture of 13 phenylthiohydantoin-amino acids. A modulated pump laser beam periodically illuminated the capillary at a point near its end. Complex deflection and diffraction effects occur at the capillary-solution interface. Perturbation of the refractive index at this interface changes the intensity of the probe beam that is measured using a small photodiode. 

The molar refractions of the amino acids were determined by measurements on their aqueous solutions and the expanded Lorenz-Lorentz equation. The refractive indices of a number of proteins were calculated from their amino acid compositions and the values for the refraction of the amino acid residues. These calculated results are in good agreement with those experimentally determined, demonstrating that refractive index is a unique characteristic of a protein. A comparison of the refractive index of heat denatured /3-lactoglobulin with the native protein demonstrated that changes in structure produced a small change in refractive index, not associated with a change in volume. 

Adair and Robinson (1) indicate that the refractive index of a protein or an amino acid is approximately determined by its elementary composition however, the structure of a molecule is also of importance. The values reported for amino acids are scattered and fragmentary (1, 10), and prior to our preliminary communication (25) no systematic investigation had accounted quantitatively for the relationship... 

In this paper, the results of a systematic study of the refractive indices of the amino acids, and some peptides and proteins, are described. The value for the refractive index of a protein calculated from the refractive increments of its amino acid residues and solution volume agrees with the experimental value and is a characteristic of the protein. The change in the refractive index of a protein as a result of denaturation has also been investigated. 

Refractive Index of Protein from Amino Acid Composition. The method used for calculating the refractive index of a protein from its amino acid composition is essentially the same as that described by Cohn and Edsall (9, Chap. 16) for calculating the specific volume of a protein from its amino acid composition. 

The importance of specific volume or density in determining refractive index is apparent in Equations 1 and 2, which are used in calculating refractive index and molar refraction. This inverse relationship between specific volume and refractive index is illustrated in Table n (cf. columns 2 and 4). The necessity of obtaining an accurate value for the specific volume of a protein in order to obtain agreement between its refractive index calculated from the amino acid composition and the determined value can be illustrated in the case of ribonu-... 

Refractive Indices of Peptides. To determine the effect of peptide formation on refractive index, the refractive indices of several peptides were determined (Table HI). The average molar refraction of water produced in peptide formation can be estimated, empirically, by subtracting the observed molar refraction of the peptide from the sum of molar refractions of its constituent amino acids. 

Effect of Ionization on the Refractive Index and Molar Refraction of Amino Acids and Proteins. Since the electrostriction produced by an amino acid does not affect its molar refraction, the ionization of an amino acid might be expected to produce no significant change in molar refraction. Table V indicates that this is the case, provided the large change in the volume of the amino acid as a result of ionization, found by Kauzmann, Bodanszky, and Rasper (23), is used in calculating molar refraction. The refractive index of an equivalent concentration of hy-... 

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