Efficacy of the peak area method of high-performance liquid chromatography (HPLC) analysis: The separation, identification, and quantification of phytochemical compounds of Moringa oleifera
Authors: Mercy Morola Oluewu  
Published: September 2024

The quantification and identification of phytochemical compounds in Moringa oleifera have received significant attention due to their potential health benefits. High-Performance Liquid Chromatography (HPLC) has become a widely used method for analyzing these phytochemicals due to its speed, adaptability, and effectiveness. However, there is a need to improve these methods to achieve more accurate quantification and understanding of these compounds. This study aims to assess the effectiveness of the peak area method in HPLC for the separation, identification, and quantification of phytochemical compounds in Moringa oleifera leaves. The main goal of the study is to use the peak area method within HPLC to measure the concentration of various phytochemical compounds in Moringa oleifera leaves. This method offers a more reliable measurement than peak height, allowing for comparison of different components' concentrations within or between samples, which is important for trace analysis in various applications. The methodology involved collecting and preparing Moringa oleifera leaves from five regions: Nigeria, Ghana, Haiti, India, and Texas, USA. The leaves were dried, ground, and extracted using methanol and ethanol. The extracts were then analyzed using HPLC, with the peak area method utilized to quantify the phytochemical compounds. The HPLC system utilized a Nucleosyl C18 column with a UV-vis diode-array detector, and the separation process was monitored at 332 nm. Key findings of the study indicated that chlorogenic acid, rutin, ferulic acid, luteolin, apigenin, and kaempferol were successfully separated, identified, and quantified from Moringa oleifera leaves using the peak area method in HPLC. Chlorogenic acid had a retention time of 15.759 minutes and a peak area of 170,844, indicating it is more polar and present in significant quantities. Rutin followed with a retention time of 16.919 minutes and a peak area of 115,599. Ferulic acid was the most abundant compound, with a peak area of 422,129 and a retention time of 17.965 minutes. Luteolin, with a peak area of 235,655 and a retention time of 19.777 minutes, was also present in considerable amounts. Apigenin and kaempferol showed retention times of 20.840 minutes and 21.039 minutes with peak areas of 343,378 and 197,585, respectively. The study also compared the extraction efficiencies of 70% ethanol and 80% methanol, finding that methanol was significantly more effective, especially for highly polar compounds like chlorogenic acid, luteolin, and rutin, with peak areas being 74% to over 1100% higher than those observed with ethanol. The study concluded that the peak area method in HPLC is a highly effective tool for the separation, identification, and quantification of phytochemical compounds in Moringa oleifera leaves. It recommended further optimization of the HPLC method and exploration of pharmacological properties of these phytochemical compounds for nutraceuticals and pharmaceuticals.