Chromatography research

1. Non-aqueous reversed phase liquid
chromatography with charged aerosol
detection for quantitative lipid analysis
with improved accuracy
Ariana Causevic, Kim Olofsson, Patrick Adlercreutz, Carl Grey
Cite: Causevic A., Olofsson K., Adlercreutz P., Grey C. (2021) Non-aqueous reversed phase liquid chromatography
with charged aerosol detection for quantitative lipid analysis with improved accuracy. Journal of Chromatography
A, 1652, 462374. https://doi.org/10.1016/j.chroma.2021.462374.

2. Abstract
There is a great need for efficient analysis of the composition of vegetable oils and
fats, since it affects the physical and technical properties. However, due to the
complex nature of these kind of samples, it is often difficult and costly. In the
present study, we developed a Non-Aqueous Reversed-Phase HPLC method that
can be used to separate and quantify different free fatty acids, fatty acid esters,
monoacylglycerides, diacylglycerides and triacylglycerides, including regioisomers
such as SOS/SSO and 1,2- and 1,3-diolein. Two 25 cm Nucleodur C18 Isis
columns in series, sub-ambient column temperature and a mobile phase gradient
composed of acetonitrile acetic acid, isopropanol and heptane were used for the
separation. The lipids were detected and quantified using a charged aerosol
detector and it was found that the peak shape highly affected the detector
response as well as the response uniformity, even when inverse gradient
compensation was employed. Thus, calibration and determination of response
factors were necessary for reliable quantification.

3. Abstract
A correlation between response factors and peak width at half peak height was
found and used for quantification of non-calibrated components. A quantification
approach was suggested including an appropriate selection of calibrated
components, depending on sample composition and the accuracy required. It was
shown in a complex oil sample that the reduced calibration approach, using only 6
instead of 33 calibrated components, resulted in virtually the same composition,
but yielded a more accurate result compared to using relative area that neglects
response factors. The method validation showed good reproducibility and
accuracy, making it an excellent tool for extensive analysis of complex lipid
mixtures.

4. Fig. 2. Representative separation of a mix of lipid standards containing FFAs, DAGs and TAGs
using NARP-HPLC with gradient elution and charged aerosol detector without inverse gradient.
Conditions: two Nuclear C18 Isis columns(150 mm x 2.1 mm I.D.,1.8 µm) connected in series.
Mobile phase gradient: See Table 1. Flow rate: 0.2 mL/min. Column temperature steps: 9°C 0-18
minutes, 15°C 18-80 minutes.

5. Table 1
System and trivial names of studies fatty acids with their abbreviations,
carbon numbers (CN) and number of double bonds (DB).