**2. Cleanup and separation of amino acids using ion-exchange chromatography**

Sample preparation is often required to improve the analysis, eliminate interference and increase sensitivity.[1] This is necessary when a sample cannot be directly analyzed or when the analysis generates poor results. Most cleanup and concentration techniques are based on separations. Separation techniques have a number of characteristics such as fractionation capacity, load capacity, adaptability to analyte volatility, type of selectivity, speed and convenience, static and dynamic procedures. The type of selectivity depends on physicochemical properties of the components in the sample. For example, differences in boiling point allow separation by distillation and are important in gas chromatographic separation, and acid/base dissociation constant is important in ion-exchange chromatographic analysis. Chromatographic separations are used both for sample preparation and core analytical separation and measurements. To check nitrogen utilization and amino acids metabolism in AM fungal symbiotic system, free amino acids from cultured tissues can be extracted with a mixture of methanol/chloroform/water, then separated on a cation exchange column (DOWEX 50 \*4-200, hydrogen form) and recovered after freeze-drying [28-30].

In the method published by Jin at al., after 3-week culture of mycorrhizal roots,[28] the extraradical mycelium (ERM) and mycorrhizal root tissues were recovered on a 38-μm sieve, rinsed with deionized water and lyophilized. The lyophilized mycorrhizal roots and ERM were ground in a mortar with a pinch of acid-washed sand and extracted with a mixture of methanol/ chloroform/water (12 : 5 : 3, v/v/v), which recovered 30–35% more amino acids than extraction with NH4HCO3 buffer (pH 8 with 0.2% NaN3) or 80% ethanol.[13]

experiment showed that NH4+ and urea are assimilated more rapidly than NO3-

increased greatly after the uptake of exogenous NH4+, urea, and NO3-

expressed in germinating spores of AM fungus *G. intraradices*.

studies of nitrogen metabolism and transport in AM fungal system.

**chromatography** 

after freeze-drying [28-30].

**2. Cleanup and separation of amino acids using ion-exchange** 

exogenous amino acids. *De novo* biosynthesis of free amino acids in the AM spores was

ratio (no exogenous glucose), the measurements of PITC-derivatized AAs with HPLC showed that Asn was the predominant amino acid in the AM spores. These results suggest that during spore germination, the main carbon source for amino acids biosynthesis is derived mostly from the degradation of stored lipids and the glyoxylate cycle. In contrast, HPLC analysis of PITC-amino acids derivatives has revealed that at a high C:N ratio (available exogenous glucose) Arg is the main amino acid produced and incorporated into the proteins of germinating AM spores[33]. This is consistent with the report of Tisserant et al [35] showing that the transcripts coding for the enzymes of Arg biosynthesis are highly

To summarize, chromatographic separation and analysis of 15N/13C-labeled amino acids have determined in what form nitrogen is taken up and assimilated, and clarified the mechanisms of Arg transport and degradation in AM fungal symbiosis. In the following sections, we will discuss in some detail the application of chromatographic methods in the

Sample preparation is often required to improve the analysis, eliminate interference and increase sensitivity.[1] This is necessary when a sample cannot be directly analyzed or when the analysis generates poor results. Most cleanup and concentration techniques are based on separations. Separation techniques have a number of characteristics such as fractionation capacity, load capacity, adaptability to analyte volatility, type of selectivity, speed and convenience, static and dynamic procedures. The type of selectivity depends on physicochemical properties of the components in the sample. For example, differences in boiling point allow separation by distillation and are important in gas chromatographic separation, and acid/base dissociation constant is important in ion-exchange chromatographic analysis. Chromatographic separations are used both for sample preparation and core analytical separation and measurements. To check nitrogen utilization and amino acids metabolism in AM fungal symbiotic system, free amino acids from cultured tissues can be extracted with a mixture of methanol/chloroform/water, then separated on a cation exchange column (DOWEX 50 \*4-200, hydrogen form) and recovered

In the method published by Jin at al., after 3-week culture of mycorrhizal roots,[28] the extraradical mycelium (ERM) and mycorrhizal root tissues were recovered on a 38-μm sieve, rinsed with deionized water and lyophilized. The lyophilized mycorrhizal roots and ERM were ground in a mortar with a pinch of acid-washed sand and extracted with a mixture of methanol/ chloroform/water (12 : 5 : 3, v/v/v), which recovered 30–35% more amino acids than extraction with NH4HCO3 buffer (pH 8 with 0.2% NaN3) or 80% ethanol.[13]

and

. In cases of a low C:N

Methylene chloride and water were added to the extraction solution to facilitate the separation of chloroform and the methanol–water phases. The methanol–water phase containing the amino acids was collected and evaporated in a rotary evaporator at 50°C Bengtsson & Odham[3] have pointed out that losses of amino acids during evaporation prior to derivatization are negligible, and, using a radioactive amino acid tracer, demonstrated low losses of amino acids co-precipitated with carbonates and hydroxides. Losses from nutrient rich samples were further reduced by acidifying the sample before evaporation. During evaporation, Maillard reaction can be avoided by keeping the temperature at 50°C and reducing evaporation pressure. A direct cation exchange has been shown to be inadequate in obtaining a sufficiently pure solution for derivative formation. However, it has been demonstrated that extraction of the aqueous sample with chloroform prior to ion exchange efficiently removes interfering organic substances without detectable losses of amino acids.[12]

Most amino acids are not soluble in nonpolar solvents and are soluble in water;[36] they display amphoteric properties (caused by COOH and NH2 groups), and many exist as zwitterions in the form R-CH(NH3+)-COO- . In acidic solutions, the amino groups are at least partly protonated whereas the ionization of the carboxyl group is very low. For the cation exchange, strong acidification is therefore necessary to convert the monoamino-acids completely to the univalent cation form. For example, at pH 2.5, 35% of Phe, 66% of Thr, and 100% of the diamino-acids are in the cationic form. In the micro-method procedure published by Bengtsson[3], the residues containing the amino acids were dissolved in 2 ml of 0.01 M HCl and loaded onto a cation exchange column, previously washed with 2 M NH4OH, deionized H2O and 2 M HCl, and followed by a wash with deionized H2O until the effluent was neutral. The neutral compounds, principally carbohydrates, were washed off the column with 5 ml of water, and the free amino acids (except cysteine(Cys) and methionine(Met), whose recoveries were low), were eluted with 2 ml of 1 M NH4OH. Sulfurcontaining amino acids are partly oxidized during the ion-exchange procedure or derivatization, therefore, this method is not suitable for recovery and purification of Cys and cystine. Nevertheless, Myung et al.[37] have developed a method employing SPME (solidphase micro-extraction) technique and GC–MS to determine homocysteine (Hcy), Cys and Met levels in aqueous samples. This method provides a new approach to the studies of S uptake and transfer in AM symbiosis.
