OPA neutralization

Figure 1. OPA neutralization by sodium bisulfite. The end product, 3, is toxic to fish.

Thus, an ideal method for OPA neutralization would be one that does not transform OPA to another toxic agent, one that does not need a solid support, and one that does not use an inconvenient agent, such as a reducing agent, and one that does not produce a dark colored solution. Our efforts in developing such an ideal, green, detoxifying neutralization method are summarized. 

One of the two major preferred OPA neutralization methods is the Cannizzaro reaction (a disproportionation reaction) catalyzed by NaOH at room temperature, done by simply mixing OPA solution with 50% NaOH at room temperature. The resulting solution showed no color changes or toxicity to fish after pH adjustment to 7. Since a Cannizzaro reaction of dialdehydes is rarely studied, three possible routes were examined as shown in Fig. 6. 

Figure 6. OPA neutralization by Cannizzaro reactions Three possible routes may lead to different end products.

Assuming both direct oxidation and Dakin reaction are possible during OPA neutralization by hydrogen peroxide, three possible products, 19, 20 and 17, could form (Fig. 11). One of them, catechol, is very toxic. 

Figure 11. OPA neutralization by hydrogen peroxide will catechol form ...

The situation is different in the case of OPA. The two aldehyde carbonyls in OPA are electron withdrawing to each other. Thus, the Dakin reaction seems unlikely to occur during OPA neutralization with hydrogen peroxide. More specifically, we expect that the first step Dakin reaction forming 16 (Fig. 13) with not occur. If the first step (the more difficult step) did occur, the second step could follow since it seemed an easier step (forming the highly toxic catechol as the main product. This is highly undesirable). 

A systematic original research of a wide range of possibilities identified two methods, which could be used safely for OPA neutralization, both meeting the needs of environmental protection and customer convenience around the world. The hydrogen peroxide approach meets the greenest demand possible. Although this work focuses on the neutralization of OPA in its 0.55% solution, with slightly modification, solid OPA (or OPA solutions with other concentrations) or compounds with similar structures should be also applicable. 

Broccoli Extraction with boiling MeOH (70%) for group separation of neutral, acid and basic AA on micro-columns Spherisorb S3 ODS2 (150x4.6mm) Phosphate buffer (0.2% NaNOjj/ACN OPA/2- mercaptoethanol UV-Vis (340 nm) [242,247]... 

OPA has been known to give a fluorescent adduct with most primary amines in the presence of a thiol compound, but only with several biogenic amines such as histidine, histamine, and glutathione in the absence of a thiol compound in a neutral or alkaline medium. In the case of histidine, it gradually reacts with OPA alone in an alkahne medium, to give a relatively stable fluorescent adduct showing excitation and emission maxima at 360 and 440 nm, respectively. Hakanson et al. optimized these reaction conditions and showed that the fluorescence intensity due to histidine reached a maximum 10 min after initiation of the reaction at pH 11.2-11.5, at 40°C. This fluorescence reaction is relatively selective for histidine and has been used in a batch method for the assay of histidine. "... 

EKC separations of aliphatic amines include electrolyte systems composed of several surfactants (SDS, cholate, Brij 35 ) modified by certain additives (urea, neutral CDs, organic solvents ), mixed CDs, and more unusual secondary phases [resorcarene-octacarboxylic acid, calixarene," poly(sodium 4-styrenesulfonate), PSSS ]. Derivatization is performed when UV (o-phthaldialdehyde, OPA, as derivatizing agents) or LIF detection [3-(2-furoyl)quinoline-2-carboxaldehyde, 5-(4,6-dichloro-s-triazin-2-ylamino)fluorescein (DTAF) as labeling agents] is designed. 

On the other hand, we revealed the mechanistic pathway of the OPA-induced fluorescence reaction of histidine, as shown in Fig. 1." In addition, we found that the fluorescent adduct of histidine rapidly forms in a neutral medium. 

OPA can be neutralized with sodium bisulfite as shown (Fig. 1). The neutralization is very fast and a colorless aldehyde-bisulfite adduct, 3, is formed. However, the product is toxic to fish even though sodium bisulfite itself is not. 

Like neutralization of glutaraldehyde, glycine neutralizes OPA, forming a fish-safe Schiff s base, 4. However, the neutralized solution is dark. Zhu et al... 

Figure 2. OP A neutralization by glycine followed by sodium borohydride reduction. The dark color of OPA-glycine adduct, 4, disappeared instantly. The disadvantage of this method is the use of a harmful reagent, sodium borohydride...

Interestingly, we found that household ammonia (diluted ammonia water solution) precipitates OPA effectively and instantly from its water solution at room temperature. However, the product mixture is toxic to fish and is not an ideal neutralization method for hospital users since qualified personnel and a filtration procedure (to remove toxic precipitate) are required. In some situations, this process may be acceptable. 

This was further confirmed by GC/MS analysis by acidifying the NaOH neutralized OPA solution to pH3 followed by immediate extraction with ethyl acetate. GC/MS shows that phthalide is the only peak present in the ethyl acetate solution. This easy ring closure is not surprising considering the fast kinetics of ring formation and the stability of the final product (Fig. 9). 

The other, preferred new method is hydrogen peroxide oxidation. Hydrogen peroxide (at different concentrations) neutralizes CIDEX OPA Solution almost instantly at room temperature (based on color reaction of OPA with glycine). No color changes are detectable after neutralization. The neutralized solution passes the fish test (this becomes an important criterion if the neutralized solution goes to rivers or oceans). 

Interestingly, many other strong oxidants, such as S% potassium persulfate and 5% sodium chlorite also neutralize OPA but at much slower reaction rates. 

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