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Application of Hydrophilic Interaction Liquid Chromatography for the Quantification of Flavonoids in Genista tinctoria Extract.

Sentkowska A, Biesaga M, Pyrzynska K - J Anal Methods Chem (2016)

Bottom Line: In the case of bare silica column more or less apparent dual retention mechanism was observed, depending on the water component content in the mobile phase.ZIC-HILIC showed better selectivity (in comparison to silica column) with the detection limit of 0.01 mg/L (only for rutin was 0.05 mg/L).Finally, this chromatographic procedure was validated and applied for the determination of some flavonoids in Genista tinctoria L. extract.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Warsaw, Pasteura 1, 02-093 Warsaw, Poland.

ABSTRACT
Hydrophilic interaction chromatography (HILIC) was employed to investigate chromatographic behavior of selected flavonoids from their different subgroups differing in polarity. Chromatographic measurements were performed on two different HILIC columns: unmodified silica (Atlantis-HILIC) and zwitterionic sulfoalkylbetaine (SeQuant ZIC-HILIC). Separation parameters such as content and type of organic modifier were studied. On ZIC column retention factors were observed to be inversely proportional to the buffer content in the mobile phase, which is the typical partitioning mechanism. In the case of bare silica column more or less apparent dual retention mechanism was observed, depending on the water component content in the mobile phase. ZIC-HILIC showed better selectivity (in comparison to silica column) with the detection limit of 0.01 mg/L (only for rutin was 0.05 mg/L). Finally, this chromatographic procedure was validated and applied for the determination of some flavonoids in Genista tinctoria L. extract.

No MeSH data available.


Related in: MedlinePlus

The retention time of rutin at different concentration of ammonium formate (95% ACN v/v pH 7).
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fig2: The retention time of rutin at different concentration of ammonium formate (95% ACN v/v pH 7).

Mentions: The higher content of both applied organic solvent in the mobile phase enhances the retention of studied flavonoids on ZIC-HILIC column. At increasing concentration of acetonitrile, water is adsorbed more strongly on the surface of the polar stationary phase. The more hydrophilic the analytes are, the more the partitioning equilibrium is shifted towards the adsorbed water layer on the stationary phase, and the more analytes are retained. It can be clearly seen on the example of rutin. For this flavonoid the retention factor increases rapidly with the increase of ACN. Its retention time is longer than 120 min, when mobile phase containing 95% of ACN is applied. Such a high value of retention factor for rutin can be shortened when ACN is replaced with MeOH (Figure 1). The layer formed onto stationary phase is less polar, when methanol is used, which is the main reason for decreasing retention of more polar compounds (myricetin, rutin, and catechin). On the other hand, methanol as a main component of the mobile phase enhances the retention of less polar flavonoids. The use of methanol as a mobile phase component reduces the retention time of the rutin but worsens the separation of other studied flavonoids. In such a situation the application of acetonitrile is necessary. However, the use of ammonium formate (pH 7.0) instead of the water (pH about 7.0) as an inorganic component of mobile phase enables shortening rutin retention time without losing the separation of other compounds (Figure 2). The biggest differences in retention times of rutin are shown on ZIC-HILIC column when 5 mM ammonium formate is used instead of water in the eluent. Shortening of the retention time from longer than 120 min (not indicated on the graph) to about 30 min is obtained. A further increase in buffer concentration also reduces the retention time of rutin but the differences are not so considerable. A similar correlation was observed for silica column, but due to the low retention of rutin on this stationary phase, the differences in retention times are insignificant. The presence of salt in the eluent shields electrostatic interactions, both attractive and repulsive. As it was reported before [25], the presence of salt in the mobile phase slightly increases the retention for hydrophobic aglycones. On the other hand, the retention factors of more polar compounds such as rutin decrease when higher concentration of buffer is applied. This fact suggests that repulsive interactions between more polar flavonoid and stationary phase surface play a key role in the retention mechanism.


Application of Hydrophilic Interaction Liquid Chromatography for the Quantification of Flavonoids in Genista tinctoria Extract.

Sentkowska A, Biesaga M, Pyrzynska K - J Anal Methods Chem (2016)

The retention time of rutin at different concentration of ammonium formate (95% ACN v/v pH 7).
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4940558&req=5

fig2: The retention time of rutin at different concentration of ammonium formate (95% ACN v/v pH 7).
Mentions: The higher content of both applied organic solvent in the mobile phase enhances the retention of studied flavonoids on ZIC-HILIC column. At increasing concentration of acetonitrile, water is adsorbed more strongly on the surface of the polar stationary phase. The more hydrophilic the analytes are, the more the partitioning equilibrium is shifted towards the adsorbed water layer on the stationary phase, and the more analytes are retained. It can be clearly seen on the example of rutin. For this flavonoid the retention factor increases rapidly with the increase of ACN. Its retention time is longer than 120 min, when mobile phase containing 95% of ACN is applied. Such a high value of retention factor for rutin can be shortened when ACN is replaced with MeOH (Figure 1). The layer formed onto stationary phase is less polar, when methanol is used, which is the main reason for decreasing retention of more polar compounds (myricetin, rutin, and catechin). On the other hand, methanol as a main component of the mobile phase enhances the retention of less polar flavonoids. The use of methanol as a mobile phase component reduces the retention time of the rutin but worsens the separation of other studied flavonoids. In such a situation the application of acetonitrile is necessary. However, the use of ammonium formate (pH 7.0) instead of the water (pH about 7.0) as an inorganic component of mobile phase enables shortening rutin retention time without losing the separation of other compounds (Figure 2). The biggest differences in retention times of rutin are shown on ZIC-HILIC column when 5 mM ammonium formate is used instead of water in the eluent. Shortening of the retention time from longer than 120 min (not indicated on the graph) to about 30 min is obtained. A further increase in buffer concentration also reduces the retention time of rutin but the differences are not so considerable. A similar correlation was observed for silica column, but due to the low retention of rutin on this stationary phase, the differences in retention times are insignificant. The presence of salt in the eluent shields electrostatic interactions, both attractive and repulsive. As it was reported before [25], the presence of salt in the mobile phase slightly increases the retention for hydrophobic aglycones. On the other hand, the retention factors of more polar compounds such as rutin decrease when higher concentration of buffer is applied. This fact suggests that repulsive interactions between more polar flavonoid and stationary phase surface play a key role in the retention mechanism.

Bottom Line: In the case of bare silica column more or less apparent dual retention mechanism was observed, depending on the water component content in the mobile phase.ZIC-HILIC showed better selectivity (in comparison to silica column) with the detection limit of 0.01 mg/L (only for rutin was 0.05 mg/L).Finally, this chromatographic procedure was validated and applied for the determination of some flavonoids in Genista tinctoria L. extract.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Warsaw, Pasteura 1, 02-093 Warsaw, Poland.

ABSTRACT
Hydrophilic interaction chromatography (HILIC) was employed to investigate chromatographic behavior of selected flavonoids from their different subgroups differing in polarity. Chromatographic measurements were performed on two different HILIC columns: unmodified silica (Atlantis-HILIC) and zwitterionic sulfoalkylbetaine (SeQuant ZIC-HILIC). Separation parameters such as content and type of organic modifier were studied. On ZIC column retention factors were observed to be inversely proportional to the buffer content in the mobile phase, which is the typical partitioning mechanism. In the case of bare silica column more or less apparent dual retention mechanism was observed, depending on the water component content in the mobile phase. ZIC-HILIC showed better selectivity (in comparison to silica column) with the detection limit of 0.01 mg/L (only for rutin was 0.05 mg/L). Finally, this chromatographic procedure was validated and applied for the determination of some flavonoids in Genista tinctoria L. extract.

No MeSH data available.


Related in: MedlinePlus