Eluents amino acid

Mixing the additive in the eluent used as a mobile phase can also modify the chromatographic system (dynamic modification), but the use of modified adsorbents has led to an improvement of resolution. Example works include that by Armstrong and Zhou [11], who used a macrocyclic antibiotic as the chiral selector for enantiomeric separations of acids, racemic drugs, and dansyl amino acid on biphenyl-bonded silica. 

Vasanits, A. and Molnar-Perl, I., Temperature, eluent flow-rate and column effects on the retention and quantitation properties of phenylthiocarbamyl derivatives of amino acids in reversed-phase high-performance liquid chromatography, J. Chromatogr., A, 832,109, 1999. 

Figure 6 Separation of amino acids on conventional ion exchange Beckman 120B Amino Acid Analyzer. Column 15 cm. Eluent 0.35 M sodium citrate buffer, pH 5.28. Flow rate 30 ml/hr. Temperature 50°C. Note that the separation requires approximately 6 hours. Compare to a modern separation shown in Figure 7. (Reproduced with permission from Beckman Instruments Fullerton, CA.)...

Crystal structures are available for many (N)4Co-amino acid complexes (Table I). Many of the diastereomers (AS, AS) in the bis-en series have been resolved using classic crystallization (usually via bromocamphor sulfonate, arsenyl-, or antimonyl-tartrate salts) or ion exchange methods (Table II). Reversed-phase ion-pair HPLC, using aryl phosphate or aryl/alkyl sulfonate ion pairing reagents in MeOH/ H20 eluent, has allowed diastereomer separations to be carried out on analytical amounts (28) (Table II). 

A universal eluent system was proposed for analysis of amino acids in biological fluids by IEC on the cation exchange resin Ostion LG ANB134. A study was carried out on the effect of the carrier pH on RP-HPLC of amines, using a Cs column and octyl sulfate as ion pairing reagent. Optimal results were obtained for the analysis of catecholamines... 

The selectivity of different stationary phase materials can be applied using columns in sequence to provide high-speed isocratic separations instead of gradient elution. An example for amino acids analysis is shown later in Figure 4.15, where the same eluent was used for all of the separations and the fraction containing the sample components of interest was switched from one column to another. 

Aspartic and glutamic acids were not retained in a buffer solution without a counter-ion however, these acids were retained by the addition of octyl sulfate to the eluent, as seen in Figure 4.12. These amino acids can form a complex with copper ions and will be retained on the stationary phase. The addition of both a counter-ion and copper ions further increased the retention and made possible... 

Figure 4.12 Effect of counter-ions and copper on the retention of amino acids. Column, octadecyl-bonded silica gel, 25 cm x 4.6 mm i.d. eluent, 0.01 M sodium acetate buffer (pH 5.6) containing 1.2 mM sodium octanesulfonate (Oc) andj or 0.1 mM copper acetate (Cu) flow rate, 1ml min-1 detection, UV 220 nm. Compounds Glu, glutamic acid, Asp, aspartic acid.

Figure 4.15 Ion-pair liquid chromatography of free amino acids using a column switching system. Column I, butyl-bonded silica gel, 50 x 4.6 mm i.d., 2, octyl-bonded silica gel, 50 x 4.6 mm i.d., and 3, octadecyl-bonded silica gel, 250 x 4.6 mm i.d. eluent, 0.01 m sodium acetate buffer (pH 5.6) containing 4 mM copper acetate and 0.8 mM sodium heptanesulfonate flow rate, 1 ml min-1 detection, UV 235 nm. Peaks 1, Tyr 2, Val 3, Met 4, His 5, Lys 6, lie, 7, Leu 8, Phe 9, Arg 10, Asp 11, Ser 12, Glu 13, Thr 14, Gly 15, Pro 16, Cys and 17, Ala. 1-9 were separated on column 1 and 10-17 were separated by a combination of columns 2 and 3.

On the other hand, optionally added co-ions of the eluent may also interfere with the ion-exchange process through competitive ion-pairing equilibria in the mobile phase. The effect of various amines added as co-ions to the polar-organic mobile phase was systematically studied by Xiong et al. [47]. While retention factors of 9-fluorenylmethoxycarbonyl (FMOC)-amino acids were indeed affected by the type of co-ion, enantioselectivities a and resolution values Rs remained nearly constant. For example, retention factors k for FMOC-Met decreased from 17.4 to 9.8 in the order... 

Peyrin, E. et al., Dansyl amino acid enantiomer separation on a teicoplanin chiral stationary phase effect of eluent pH, J. Chromatogr. A, 923, 37, 2001. 

The eluent front was marked on the glass plates prior to drying over a hot plate. Amino acids were visualized as purple spots by application of the ninhydrin spray reagent to the plate and drying with hot air. Amino acids with zero mobility (Rf = 0 in BUOH/HAC/H2O) are assumed to be cross-links (Keller et al., 1984). 

Part of the high molecular weight fractions contained ninhydrin-positive compounds displaying Rf 0 on TLC-plates (BUOH/HAC/H2O), characteristic of cross-links. To remove contaminating common amino acids, the selected fractions were further purified by adsorption chromatography on cellulose. Most of the amino acids eluted with BUOH/HAC/H2O, accounting for about three quarters of the total reactivity towards ninhydrin. The amino acids eluted with BUOH/HAC/H2O displayed spots with widely varying mobilities after TLC separation with the same eluent, whereas those eluted with water showed zero and very low mobility. 

The retention of polar amino acids can be enhanced by the inclusion of decyl sulfate (203) or other anionic surfactants (234) in the eluent. The use of such agents in the mobile phase modifies stationary phase interactions... 

Fig. 19. Efficiency of RPC in protein separation. Isocratic RPC separation of murine epidermal growth factor (after elution as a single peak from a diethylaminoethyl cellulose column). Column RP 18 (300x7.8 mm dP = 10 pm) 40 °C eluent 0.04 M triethylamine acetate in water — acetonitrile (74 26), pH 5.6 flow rate 0.8 ml/min UV detection at 254 nm, 0.05 AUFS injection 100 pg in 100 pi. The a peak was due to the polypeptide of 53 amino acid residues (M =6040 g/mol), the p component did not contain the asparagine residue in position 1. (From Ref.77) with permission)...

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. 

Fig. 11 Separation of amino acid standards derivatized with PITC. Eluent A sodium acetate/triethy-larnine buffer (pH 6.4) eluent B 60/40 acetonitrile/water. Pico-Tag column. Peak identification 1 Asp, 2 Glu, 3 Ser, 4 Gly, 5 His, 6 Arg, 7 Thr, 8 Ala, 9 Pro, 10 ammonia, 11 Tyr, 12 Val, 13 Met, 14 Cys, 15 lie, 16 Leu, 17 Phe, 18 Lys. (From Ref. 184. Copyright 1984 Elsevier Science.)...

YP Belov, AY Aksinenko, B Blessington, AH Newman. Chiral aminophosphonate eluent for enantiomeric analysis of amino acids. Chirality 8 122-125, 1996. 

C van Wandelen, SA Cohen. Using quaternary high-performance liquid chromatography eluent systems for separating 6-aminoquinolyl-V-hydroxysuccinimidyl carbamate-derivatized amino acid mixtures. J Chromatogr A 763 11-22, 1997. 

---