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Logo of mexGuide for AuthorsAboutExplore this JournalMethodsX
MethodsX. 2017; 4: 256–259.
Published online 2017 July 19. doi:  10.1016/j.mex.2017.07.002
PMCID: PMC5547239

Analysis of biodiesel by high performance liquid chromatography using refractive index detector

Graphical abstract

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Keywords: HPLC, Biodiesel, Refractive index, UV detector


High-performance liquid chromatography (HPLC) was used for the determination of compounds occurring during the production of biodiesel from karanja and jatropha oil. Methanol was used for fast monitoring of conversion of karanja and jatropha oil triacylglycerols to fatty acid methyl esters and for quantitation of residual triacylglycerols (TGs), in the final biodiesel product. The individual sample compounds were identified using HPLC. Analysis of fatty acid methyl esters (FAMES) in blends of biodiesel by HPLC using a refractive index and a UV detector at 238 nm. Individual triacylglycerols, diacylglycerols, monoacylglycerols and methyl esters of oleic, linoleic and linolenic acids and free fatty acids were separated within 40 min. Hence HPLC was found to be best for the analysis of biodiesel. Analysis of biodiesel by HPLC using RID detector. Estimation of amount of FAMES in biodiesel. Individual triacylglycerols, diacylglycerols, monoacylglycerols and methyl esters of oleic, linoleic and linolenic acids and free fatty acids were separated within 40 min.


The standards (mixture of methyl esters) were purchased from Sigma-Aldrich Chemicals Pvt. Ltd, India.

Method details

Jatropha and Karanja oil characterisitics

The non-edible crude jatropha and karanja oils were purchased from local market and stored at 4 °C to avoid rancidity of the vegetable oil. Major fatty acid profiles of jatropha and karanja oils were given in Table 1. The characteristics of jatropha and karanja oils were determined according to the standard methods given in Table 1.

Table 1
Fatty acid profile of Jatropha and Karanja oil.

Lipase enzyme – biocatalyst

The following lipases were procured from Sigma – Aldrich Chemicals Pvt. Ltd., Bangalore: and other chemicals were listed in Table 2

  • Pseudomonas fluorescens lipase (300 U/mg)
  • Candida rugosa lipase (700 U/mg)
  • Rhizopus arrihizus lipase (10.5 U/mg)
  • Aspergillus oryzae lipase (15.5 U/mg)
  • Candida antartica lipase (600 U/mg)
Table 2
Specifications of chemicals employed in the investigation.

HPLC analysis for biodiesel

A High Performance Liquid Chromatographic system (HPLC Model- LC 20 AT Prominence, Shimadzu, Japan) fitted with Refractive index detector (RID-10A, Shimadzu, Japan) and millennium 32 system software was used to quantify the fatty acid methyl esters produced during reaction. Separations were carried out on a 238 nm in Luna C18 column of particle size 5 mm and (250 × 4.6 mm) I.D. Methanol mobile phase was filtered through a 0.45 μm membrane filter (Millipore), and then degassed ultrasonically prior to use. The flow rate was 1 mL min−1, the injection volume was 20 μL and the column oven temperature was maintained at 40 °C. Each component in the samples analyzed was identified by comparing its retention time with that of the respective standards. Quantification was carried out by integration of the peaks using external standards followed by calculating the% yield as weight of methyl esters produced to weight of oil initially taken [1].

Preparation of standard

A stock solution was prepared by dissolving 40 mg of standard in 2 mL of methanol. Five serial dilutions were made from the stock solution namely 2, 4, 8, 12 and 16 mg/ml using methanol as solvent. The base line for HPLC system was set according to the program mentioned in the analysis section. Once the base line was set, the samples from the stock solution were analyzed by injecting 20 μL and the corresponding peak area values are noted. The particular methyl ester peaks are identified by taking the retention time as reference. The calibration charts were drawn for the values of peak area obtained vs concentration of the sample for each methyl esters individually as shown in Fig. 1.

Fig. 1
Standard graph of biodiesel using refractive index detector.

Additional information

HPLC was applied for the analysis of biodiesel than GC analysis. LC was found to be operationally superior to GC because of the aforementioned reasons, and it was directly applicable to most biodiesel fuels [2]. Simultaneous determination of aromatic compounds and FAME in blends of biodiesel with petrodiesel by HPLC using a refractive index and a UV detector [3]. It has been used to identify various components of biodiesel mixtures including fatty acid methyl esters, triglycerides, diglycerides, monoglycerides, and fatty acids, among others. HPLC analysis time is generally shorter than GC, and no derivitization step is needed. Additionally, lower analysis temperatures allow for the use of standard columns [4].


1. Bakkiyaraj S., Syed M.B., Devanesan M.G., Thangavelu V. Production and optimization of biodiesel using mixed immobilized biocatalysts in packed bed reactor. Environ. Sci. Pollut. Res. 2015 [PubMed]
2. Foglia T.A., Jones K.C., Nuñez A., Phillips J.G., Mittelbach M. Comparison of chromatographic methods for the determination of bound glycerol in biodiesel. Chromatographia. 2004;60:305–311.
3. Kami&z.hfl;ski M., Gilgenast E., Przyjazny A., Romanik G. Procedure for and results of simultaneous determination of aromatic hydrocarbons and fatty acid methyl esters in diesel fuels by high-performance liquid chromatography. J. Chromatogr. A. 2006;1122:153–160. [PubMed]
4. Monteiro M.R., Ambrozin A.R.P., Lião L.M., Ferreira A.G. Critical review on analytical methods for biodiesel characterization. Talanta. 2008;77:593–605.

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