Page 69 - Application Handbook - Liquid Chromatography

Application No.L492A
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n Comprehensive Separation of Triglycerides in partition number in the second dimension.
Borage Oil with LCMS-IT-TOF Detection Use of the LCMS-IT-TOF as the detector for precise mass
As mentioned above, separation in the first dimension in this measurement in the second dimension permits detailed
analysis is conducted based on the presence or absence or the qualitative analysis of the many components eluted following
difference in the number of double bonds in the fatty acid separation by the Nexera-e. The mass spectra corresponding
side chain. When a silver ion column is used, the greater the to the white-circled peaks A, B and C of Fig. 2 are shown in
number of double bonds in the triglyceride, the stronger the the lower part of Fig. 2. The structural information was
retention will be. However, it is also possible that retention obtained from the peak of diglyceride with one side chain
will be affected depending on the positions of the double detached, and the triglyceride structure were determined as
bonds or the side chain length. In the second-dimension follows:
reversed phase separation, two-solution gradient elution is A : POP
adopted in which, due to the high hydrophobicity of B : OOP
triglycerides, neither water nor buffer solution, etc., is used, C : PγLnP
but a non-aqueous organic solvent is used for the mobile Where,
phase. With this separation mode, elution tends to proceed in P : Palmitic acid
the order obtained by subtracting twice the number of O : Oleic acid
double bonds from the total number of triglyceride carbon γLn : γ-Linolenic acid
atoms, which is referred to as the partition number. The top Since these compounds each have one to three double
portion of Fig. 2 shows a two-dimensional plot drawn based bonds, they are eluted from the first-dimension column
on the output of the LCMS-IT-TOF mass spectrometer. To in this order. As lipid-related compounds often display
facilitate identification of triglycerides using the order of no UV absorption, and gradient elution cannot be
elution described above, a grid is drawn superimposed on the applied with differential refractive index detection, a
plot. From this plot, it can be seen how separation is combination of the Nexera-e and ELSD, or a triple
conducted according to the difference in the number of quadrupole or LCMS-IT-TOF mass spectrometer can be
double bonds in the first dimension, and the difference in considered essential for exhaustive analysis in this field.

PN : carbon number
NDB : number of double bonds

A B

C

PO ЍLnP
PO
POP OO OOP PP PЍLnP
DB = 1 DB = 2 DB = 3
[M+H]
+
PP

Fig. 2 Comprehensive 2D Plot of Triglycerides in Borage Oil with LCMS-IT-TOF in Addition to the Mass Spectra of Assigned Blobs

Data provided by University of Messina Prof. Luigi Mondello and Chromaleont S.r.l.

Second Edition: Feb. 2016
First Edition: May. 2015

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