HPLC
Applications
note
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HPLCAnalysis ofVitamins
Themarket for nutritional and dietary supplements is growing
tremendously.And, as these nutraceutical and dietary supple-
ment markets grow, the need for simple, rugged, and accurate
high performance liquid chromatography (HPLC)methods to
analyze vitamins becomes increasingly important.
Vitamins encompass awide range of organic compounds. They
are required by the human body in onlyminute quantities.Yet,
they have significant impact at the cellular level. Vitamins play
a vital role in converting fat and carbohydrates to energy, regu-
latingmetabolism, and other bodily functions.
Vitamins can be broadly classified into two groups, water-solu-
ble vitamins and fat-soluble vitamins. Fat-soluble vitamins
include such constituents asVitaminA(retinol) andVitaminD3
(cholecalciferol). VitaminAhas been found to play an important
role in proper growth and eye function, whileVitaminD is
required for proper bone and tooth growth.
Fat-SolubleVitamins
TheRestekUltraC18 column is an ideal first choice to separate
awide range of fat-soluble vitamins. Its very retentive, high-
purity packing exhibits excellent peak shape. The silica has a
carbon load of 20% and is fully end-capped, which eliminates
any unwanted silanol interactions and improves column-to-col-
umn reproducibility.
The fat-soluble vitamins are extremely hydrophobic andmust
be dissolved in an organic solvent.Amixture of six fat-soluble
vitaminswere separated using anUltraC18 column (Figure 1).
The analyteswere dissolved in diethyl ether. The sample solu-
tionwas then analyzed using a simple isocraticmobile phase
(acetonitrile:methanol [90:10]) with a stepped flow rate
(1mL/min. from 1minute to 5minutes, then 2mL/min. from 5
minutes to 50minutes).All six constituents are easily resolved
and show excellent peak shape.
Water-SolubleVitamins
The group of compounds known aswater-soluble vitamins is
very diverse chemically, including both basic and acidicmole-
cules. Some of thewater-soluble vitamins, such as thiamin and
ascorbic acid, are very polar and thus difficult to retain by
reversed phaseHPLC.Many reversed phasemethods for water-
soluble vitamins require ion-pairing reagents in order to retain
themore polar analytes. However, theUltraAqueous C18 col-
umn easily separates sixwater-soluble vitaminswith a gradient
elution and relatively simplemobile phases that contain no ion
pairing reagents (Figure 2).
Figure 2
TheUltraAqueous C18 column easily separates six
water-soluble vitamins.
Figure 1
TheUltraC18 column provides excellent peak shape and
resolution of fat-soluble vitamins.
LC_0144
PeakList:
Conc.:
(mg/mL)
1. solvent front
2. menadione (vitaminK3)
0.45
3. all-trans-retinol (vitaminA) 0.34
4. vitaminD3
0.4
5. unknown
6. alpha tocopherol (vitaminE) 2.4
7. alpha tocopherol acetate
(vitaminEAcetate)
2.4
8. unknown
9. phylloquinone (vitaminK1) 0.84
Column:
UltraC18
Catalog #:
9174575
Dimensions: 250 x 4.6mm
Particle Size: 5µm
Pore Size:
100Å
Sample:
Inj.:
1µL
Solvent:
all analytes dissolved
inHPLC-grade
diethylether.
Conditions:
Mobile phase: acetonitrile:
methanol (90:10)
Time (min) Flow (mL/min)
1.00
1.00
5.00
1.00
5.01
2.00
50.00
2.00
Temp.:
30°C
Det.:
UV@ 280nm
LC_0141
PeakList:
Conc.
(µg/mL)
1. thiamin (B1)*
250
2. ascorbic acid (C)
1,000
3. unknown
n/a
4. nicotinic acid (B3)
1,000
5. unknown
n/a
6. pantothenic acid (B5) 1,000,000
7. folic acid (B9)
500
8. riboflavin (B2)*
250
9. methyl paraben (preservative) 0.2
*Initial dilutions of B1 andB2
basifiedwith ammonium hydroxide.
Column:
UltraAqueousC18
Catalog #:
9178575
Dimensions: 250 x 4.6mm
Particle size: 5µm
Pore size:
100Å
Sample:
Inj.:
1µL
Solvent:
all analytes dissolved
inHPLC-gradewater
Conditions:
Mobile phase: A: 25mM potassium
phosphate, pH 2.00:
methanol (95:5)
B: methanol:25mM
potassium phosphate,
pH 3.5 (60:40)
Hold 0%B: 0-6min.
Step to 25%B: 6.01min.
25-100%B: 6.01-11min.
Hold 100%B: 11-16min.
Flow:
1.0mL/min.
Temp.:
27°C
Det.:
UV@ 254nm
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