| Since high performance liquid chromatography (HPLC) has been used widely in medicine and pharmacy, its qualitative and quantitative accuracy is of very importance for analysis works, such as diagnosis, drug development and quality control. However, metal chelation effect may make serious peak tailing even new impurity peaks, and it also may change peak profile, area, and reproducibility at the end. All of that were interferefering for accuracy of chromatography. Although metal chelation effect was described in some papers, hardly research on quantitative-effect relationship has been shown. In this paper, simple mode compound was used to demonstrate such a topic. The case of encounter between analyst and metal ion was simulated by quantitatively adding metal ion into sample solution. Then chromatographic parameters (mainly peak symmetry) were calculated to evaluate the subsequent change of chromatographic behavior. So that the influence of metal ion in system on peak profile and change of chromatographic behavior were quantized, and what's more, the possible avoidance and resolving method was discussed. The study was an attempt to supply reference for application of HPLC.Citric acid and 2,2'-dipyridyl were chosen as mode compound. Citric acid is a representative organic acid in plant drugs, with simple structure and clear chelating sites. Come to chelating 2,2'-dipyridyl, it could indicate the case that compound is effected by metal ion under basic environment. Besides, it could be compared with 4, 4'-dipyridyl, which reserves similar structure but non-chelating property, under same operation. The experiment was designed as following way:①Experience-based sequential optimization method was adopted to build optimal HPLC analysis condition for the samples;②Quantization measure, mainly based on peak symmetry, was constructed to evaluate the change of peak after adding metal ions with various species and amount into citric acid and 2,2'-dipyridyl solution respectively;③The analysis of analyst-metal mixture was performed again under pH-changed eluent. Under acidic eluent condition, citric acid was analyzed after adding metal ions with various species and amount; And under basic eluent condition, 2,2'-dipyridyl and 4,4'-dipyridyl was analyzed after operation similar to citric acid; the later was control sample;④Protecting effect of EDTA(disodium salt)was tested as sample-additive and as eluent-additive respectively.Through above experiments, following facts were discovered:①Influence of metal ion on HPLC analysis of chelating compound1)For chelating compound, metal ion remarkably effects its peak profile, causes deterioration of peak symmetry(tailing), and even one to several impurities peaks; Existence of partial organic solvent show few effect on interaction between analyst and metal ion; influence of metal ion was proportional to its amount in solution;2)Different chelating activity and interaction rate may effect the reproducibility of chromatography. While chromatography of 2,2'-dipyridyl showed a good reproducibility among first injection, 24h and 48h, that of citric-acid-metal-ion mixture changed much after 24h.3)As difference of chelating activity and stability, effects of different metal ion were various. For citric acid:FeⅢ>FeⅡ>AlⅢ>ZnⅡ≈CaⅡ;for 2,2'-dipyridyl:FeⅡ>FeⅢ>AlⅢ>CaⅡ>ZnⅡin pH7.0 eluent;FeⅡ>FeⅢ>CaⅡ>ZnⅡ>AlⅢin pH8.0 eluent.4)pH of eluent could change interaction much between analyst and metal ion. Acidic eluent restrained chelation between citric acid and metal ion, especially pH2.0; and basic eluent enhanced chelation between 2,2'-dipyridyl and metal ion, especially pH9.0. It was accordant to conclusion of inorganic chemistry that chelating interaction was inhibited in acidic environment and promoted in basic environment.②Efficiency of EDTA as protective agent1)Adding EDTA into citric acid solution could not avoid interaction between analyst and metal ion, instead, made analysis result unacceptable; Whereas, Adding EDTA into 2,2'-dipyridyl solution could protect analyst, making peak profile keep well to some extend.2)When EDTA was used as inorganic-phase in eluent to analyze citric acid, peak widening, height falling and baseline rising, etc, appeared in chromatogram. There was not expected result. When it comes to 2,2'-dipyridyl, peak profile and symmetry were good and keep stable even after adding metal ion.3)Combining changing degree and peak profile, it is better to used EDTA as eluent-additive than as sample-additive. According to above facts, as protecting agent in HPLC analysis, EDTA is not efficient enough for all compounds.
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