Page 4 - Application Notebook - Solution for Food Development

LAAN-A-LC-E236

Application High Performance Liquid Chromatography

News Simultaneous Analysis of Water-Soluble Vitamins by

Newly Developed "Shim-pack MAqC-ODS Ⅰ" Column
No.L459

Vitamins play vital roles in the metabolic activities of the n Analysis of Standard Solution
body. However, because they cannot be synthesized in A standard solution was prepared following the
sufficient quantities in the body, if at all, they must be procedure outlined in Fig. 1. As folic acid, riboflavin,
ingested either in our meals or by other means, such as and biotin are only slightly soluble in water, they were
tablets.
Vitamins can be classified into two groups, water- first dissolved in dilute alkali solution, and then
soluble and fat-soluble, and their analysis is generally dissolved in 10 mmol/L phosphate buffer (sodium)
conducted by high-performance liquid chromatography. solution (pH 2.6). The standard solution was adjusted
Of the two types of vitamins, the water-soluble type to obtain concentrations of 20 mg/L each.
consists mainly of highly polar basic components which An example of analysis using the Shim-pack MAqC-
exhibit weak retention in analysis by reversed-phase ODS Ⅰ is shown in Fig. 2, and the analytical conditions
liquid chromatography. Since they exhibit weak used are shown in Table 1. For comparison, analysis
retention, the analysis is typically conducted using ion- was conducted using a typical ODS column with mobile
pair reagents. phase that was spiked with an ion-pair reagent. Those
Using ion-pair reagents can be challenging because of analysis results are shown in Fig. 3, and the analytical
the longer analysis time, widening of slow eluting conditions are shown in Table 2.
peaks, difficulty in obtaining sufficient sensitivity and The results using the Shim-pack MAqC-ODS Ⅰ
long equilibration times. Analysis efficiency is also demonstrate that the analysis time can be shortened
compromised due to additional work-flow due to the use of gradient analysis. Furthermore, even
requirements, such as mobile phase preparation and the late eluting substances show sharp peaks.
column conditioning.
Here, we introduce an example in which the newly
developed "Shim-pack MAqC-ODS Ⅰ" column is used
to conduct simultaneous analysis of water-soluble
Folic acid
vitamins without the addition of ion-pair reagents to Thiamine (Vitamin B1)* Riboflavin (Vitamin B2)
Niacin
the mobile phase. Nicotinamide Biotin
Pyridoxine (Vitamin B6)*
n Overview of Shim-pack MAqC-ODS Ⅰ Ca Pantothenate
The Shim-pack MAqC-ODS Ⅰ column is a reversed- 10 mmol/L 20 mmol/L
Aqueous sodium
phase column packed with a silica gel containing metal Phosphate hydroxide
and an added octadecylsilyl (ODS) group. In addition to (sodium) buffer
the hydrophobic interaction effect provided by the Standard stock solution
ODS, the metal content effectively contributes by (500 mg/L each) Standard stock solution
(500 mg/L each)
providing cation-exchange effects. This enhances the
retention of basic compounds, thereby permitting
analysis using a conventional buffer instead of an ion- 10 mmol/L
Phosphate
pair reagent such as sodium alkyl sulfonate (e.g. sodium (sodium) buffer
1-hexanesulfonate), which is required when conducting Standard solution
ion-pair chromatography. (20 mg/L each)
It is difficult to improve throughput in ion-pair
chromatography due to the time required for *Hydrochloride was used for substances indicated with an asterisk.
reequilibration following gradient elution. While on the
other hand, in isocratic elution, sensitivity is Fig. 1 Standard Solution Preparation
compromised due to peak broadening from slow
eluting substances. Thus, its application to
simultaneous analysis is not always appropriate.
Use of the Shim-pack MAqC-ODS Ⅰ permits the
retention of highly polar basic compounds without the
use of ion-pair reagents, thereby making it possible to
apply gradient elution. As a result, simultaneous
analysis of highly polar basic compounds and other
components can be achieved while shortening the
analysis time and improving sensitivity.
These features demonstrate the powerful advantages
of this column for simultaneous analysis of water-
soluble vitamins, as well as impurities in
pharmaceutical products that contain large quantities
of basic compounds.