Determination of main compound classes in shea butter using size exclusion chromatography and refractive index detection

Abstract

Introduction: Shea butter is a kernel derived fat which includes several classes of compounds including lipids like tri- and diacylglycerols as well as unsaponifiable components.
Background: Accurate determination of the compound class composition of shea butter is important for industry to quantify the levels of wanted compounds. Size exclusion chromatography (SEC) coupled with refractive index detection (RID) can be utilized to separate and quantify lipids based on their molecular size.
Aim(s): The aim of the project was to establish and validate as SEC-RID method for quantifying compound class distributions in shea butter. The project aimed to determine the content of triacylglycerols (TAGs), diacylglycerols (DAGs), karitene and the combined amount of monoacylglycerols, free fatty acids and unsaponifiable compounds. An additional aim was evaluating the viability of using charged aerosol detection (CAD) for the analysis of shea butter by comparing the response factors of coeluting shea analytes.
Methods: The primary method used for the project was SEC coupled with RID. The method employed a Styragel HR2 column connected in series with two Styragel HR1 columns. To optimize separation, parameters such as flow rate, temperature and injection volume were varied. Validation of the method was planned following the Codex Alimentarius guidelines for fats in food products. A CAD was also utilized.
Results: The method was unable to achieve the resolution necessary to separate the TAGs and DAGs. Significant band broadening was found to originate from the columns. The calculated response factors of standard solutions using CAD showed differences in response for some of the coeluting analytes. Large confidence intervals were observed for several standards which decrease the reliability of the result.
Conclusion: Measures taken to increase resolution were unsuccessful and the method was therefore not validated. Differences in response factor in CAD indicate that calibration of an entire analyte class is not possible.

Popular Abstract

Shea butter is a natural fat obtained from the kernels of the African shea fruit. It is valued for its wide range of uses, from food products to pharmaceuticals and cosmetics. Shea butter is made up of many types of compounds which can vary in abundance between different produced butters. Fatty molecules known as triacylglycerols (TAGs) constitute the largest fraction of shea butter with other fats called diacylglycerols (DAGs) and monoacylglycerols (MAGs) being present in lower amounts. Shea butter is also high in unsaponifiable content in comparison with other seed oils. Unsaponifiable compounds are molecules found in fats which do not form soap when they are exposed to a strong base. Because different industries require different compositions of shea butter, it is important to determine how much of each compound class is present. These compound classes vary in molecular size, which this study exploits by using an analytical technique called size exclusion chromatography (SEC). SEC uses columns filled with a porous material with pores that the analytes can move in to. The pores in the column are of different sizes, which means smaller molecules will have a larger volume available to move in. When a sample is run through the column, smaller molecules will therefore stay in the porous material for a longer time which creates a separation between the molecular classes. The amount of each class can then be measured using a detector. In this study a refractive index detector (RID) was used for this measurement. A RID works by measuring the difference in how much light is scattered as the different compounds flow through it, indicating their presence and concentration. Issues arouse when using the method as all the compound classes were not separated as intended. The columns could not separate the TAG and DAG compounds which meant their individual amounts could not be determined. Many measures were done to resolve this issue, including changing methods parameters like flow rate and temperature as well as changes to the equipment. These changes did not have a significant impact on the separation. Solvent was also run backwards through the columns as it was theorized that compounds could have gotten stuck in the pores, thereby blocking parts of the available column volume. This measure increased the separation but not enough to separate the TAGs and DAGs. The two groups could not be separated and as a result a validation of the method was never performed.

• author: Ulvenlund, Albert
• supervisor: Margareta Sandahl; Azemina Bajramova
• organization: Centre for Analysis and Synthesis
• course: KASM01 20252
• year: 2026
• type: H2 - Master's Degree (Two Years)
• subject: Chemistry
• keywords: Size exclusion chromatography, Shea butter, Refractive index detection, Band-broadening, Charged aerosol detection, Analytical chemistry
• language: English
• id: 9222809
• date added to LUP: 2026-03-02 11:13:41
• date last changed: 2026-03-02 11:13:41