Page 29 - Application Notebook - Solution for Food Development

LAAN-A-LC-E269A

Application High Performance Liquid Chromatography

News Comprehensive 2D Separation of Triglycerides in

Vegetable Oil with ELSD/LCMS-IT-TOF Detection
No.L492A

Triglycerides, molecules consisting of a glycerol backbone to which acid, oleic acid, and palmitic acid, it offers a variety of health
three fatty acids are attached via ester bonds, are considered effects associated with these substances, such as moisturizing
important functional components in both animal oil and vegetable effect, wrinkle prevention, etc. Compared with other vegetable
oil. Triglycerides display low solubility in aqueous solvents, and oils, Borage oil is rich in γ-linolenic acid, which is said to be
their separation has typically been conducted by either silver ion- effective in maintaining female hormonal balance. Triglycerides
mediated normal phase analysis or reversed phase analysis using in natural fats and oils are generally characterized by the lengths
an organic solvent. However, as there are numerous molecular of their alkyl chains and the number and positions of the double
species consisting of combinations of fatty acids, mutual bonds in the alkyl chains. Triglycerides having double bonds in
separation of the triglycerides in natural fats can be difficult using particular are said to possess antioxidant action, and there is
any single set of separation conditions. The Nexera-e considerable demand for the separation of these triglycerides
comprehensive two-dimensional liquid chromatograph effectively depending on the presence or absence of double bonds. It is
achieves mutual separation of such complex components. known that strong interaction is displayed by the formation of a
When conducting comprehensive two-dimensional liquid complex comprising the double bond of an alkyl chain with a
chromatography, different separation modes are generally selected silver ion. Utilizing this property, an HPLC method in which a
for the first and second-dimension separations, and depending on stationary phase impregnated with silver is relatively often used
the differences in separation selectivity between these dimensions, to achieve selective retention of compounds containing double
improved separation is typically seen for components that are bonds. Here, using the Nexera-e to achieve comprehensive
difficult to separate in a single, one-dimensional analysis. Here, separation of multiple components, a silver ion column (normal
using borage oil as a sample that contains many triglycerides, phase conditions) with strong retention for double bonds was
micro-scale separation was conducted in the first separation using used for the first-dimension separation, an ultra-high-speed
a silver column (normal phase conditions), and reversed phase reversed phase analytical column was used for the second-
separation was conducted in the second dimension by using a dimension separation, and an ELSD was used for detection.
two-liquid gradient with non-aqueous organic solvents. Detection The ELSD converts the target compound to fine particles by
was conducted using a combination of an evaporative light evaporating the eluent exiting the column, and by measuring
scattering detector (ELSD) and an ion trap time-of-flight mass the scattered light, triglycerides, which display almost no UV
spectrometer (LCMS-IT-TOF). The analytical conditions are shown absorption, are effectively detected. Fig. 1 shows a
in Table 1. comprehensive two-dimensional representation of the
separation pattern (horizontal axis: separation in the first
n Comprehensive Separation of Triglycerides in dimension with a silver ion column × vertical axis: reversed
Borage Oil with ELSD Detection phase separation in the second dimension) generated using the
Borage oil is a vegetable oil that is obtained from the seeds of specialized two-dimensional analysis software, ChromSquare.
Borago officinalis, an annual herb. Rich in triglycerides The use of comprehensive two-dimensional separation
containing such fatty acid chains as linoleic acid, γ-linolenic permitted difficult-to-achieve high separation using a single set
of separation conditions, by which thirty-seven of the elution
Table 1 Analytical Conditions
peaks were confirmed.
[Column1] : Ag custom column (150 × 1.0 mm; 5.0 µm)
Mobile Phase : A; 1.5 % v/v of Butyronitrile in n-Hexane
B; 2.4 % v/v of Butyronitrile in n-Hexane
Time Program : B Conc. 0 % (0 min) → 100 % (40 min) → 100 % (150 min)
Flowrate : 0.007 mL/min (split)
Column Temp. : 30 °C
Injection Volume : 2 µL
Modulation Time : 1.5 min
[Column2] : Ascentis Express C18 column
(Supelco, 50 × 4.6 mm; 2.7 µm)
Mobile Phase : A; Acetonitrile
B; Isopropanol
Time Program : B Conc. 30 % (0 min) → 30 % (0.08 min) → 40 % (0.1 min)
→ 70 % (1.2 min) → 30 % (1.21 min) → 30 % (1.5 min)
Detector : Shimadzu ELSD LT-Ⅱ
Flowrate : 4 mL/min
Evaporative Temperature : 58 °C
Nebulizing Gas Pressure : 260 kPa
Detector : LCMS-IT-TOF
Flowrate : 2 mL/min from the 2D pump was split to
0.8 mL/min prior entering the APCI probe.
[MS Conditions]
Ionization Mode : APCI positive
Nebulizer Gas Flow : 2.0 L/min
Interface Temperature : 400 °C
Block Heater Temperature : 230 °C
CDL Temperature : 230 °C
Scan : m/z 300-1200 Fig. 1 Comprehensive 2D Plot of Triglycerides in Borage Oil with
ELSD Detection