Page 68 - Application Handbook - Liquid Chromatography
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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
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