Vitamins

Vitamins are essential components for maintaining physiological functions in living bodies. In particular, five water-soluble essential vitamins, B1, B2, C, nicotinic acid and nicotinamide are sometimes lacking in human bodies. The necessary quantities of three water-soluble vitamins, B6, B12 and folic acid have been determined by the NRC (National Research Council), though these vitamins are seldom lacking in human bodies. This chromatogram shows an example of the analysis of such water-soluble vitamins except folic acid. This analysis has been performed by reversed phase mode with gradient elution. Good calibration curves are obtained for the vitamins using RSpak DE-413 (a column for reversed phase chromatography). As an example, calibration curves for vitamin B2 and vitamin B6 are shown below.

Sample : 20micro-L
1. Vitamin C
2. Niacin
3. Vitamin B6
4. Niacinamide
5. Vitamin B1
6. Vitamin B12
7. Vitamin B2

Column       : Shodex RSpak DE-413 (4.6mmID*150mm)
Eluent       : (A); 1/30M Potasium phosphate buffer(pH6.8)/CH3CN=98/2
               (B); H2O/CH3CN=50/50
               0min to 4 min, 100% (A) 
               4min to 15 min, 100% (A) to 30% (A)
               after 15 min, 30%(A) 
Flow rate    : 0.6mL/min
Detector     : UV(254nm)
Column temp. : 35deg-C

Compared with the analysis using an ion pair reagent, isocratic analysis can be easily carried out by using the RSpak NN-814 column with a buffer eluent containing some organic solvent. This is a new analysis method, which does not require an ion pair reagent. The NN-814 is a polymer-based reversed phase column containing a small amount of sulfo groups.

Sample :
1. Pantothenic acid
2. Orotic acid (Vitamin B13)
3. Pyridoxine HCl (Vitamin B6)
4. Nicotinamide
5. Vitamin B1
6. Vitamin B2

Column       : Shodex RSpak NN-814 (8.0mmID*250mm)
Eluent       : 45mM KH2PO4 + 55mM Na2HPO4 + 7% CH3OH
Flow rate    : 1.0mL/min
Detector     : UV(210nm)
Column temp. : Room temp.

Six vitamines and caffein were separated isocratically using RSpak DM-614.

Sample :
1. Vitamin C
2. Vitamin B13
3. Vitamin B6
4. Vitamin B1
5. Vitamin B3
6. Caffeine

Column       : Shodex RSpak DM-614 (6.0mmID*150mm)
Eluent       : 0.055M Na2HPO4 + 0.045M KH2PO4 aq.
Flow rate    : 1.0mL/min
Detector     : UV(254nm)
Column temp. : 28deg-C

RSpak NN-614 is an improved version of OHpak M-414, which was recommended by a Japanese standard, “Methods of Analysis in Health Science” (2005). The total quantity of vitamin B1 was measured using RSpak NN-614 with a phosphate buffer eluent. The vitamin B1 separated by the column is converted into thiochrome using a postcolumn reagent and detected by a fluorescence detector.

Sample : Thiamine hydrochloride

Column       : Shodex RSpak NN-614 (6.0mmID*150mm)
Eluent       : 45mM KH2PO4 + 55mM Na2HPO4 aq.
Reagent      : 0.01% K3Fe(CN)6 + 15% NaOH
Flow rate    : (Eluent); 0.7mL/min, (Reagent); 0.7mL/min
Detector     : Fluorescence detector(Ex.: 375nm, Em.: 450nm)
Column temp. : 25deg-C

This shows the analysis of vitamin B2 used with AFpak AAF-894, an affinity chromatography column. Affinity chromatography is a method suitable for fractionation and purification of a very small amount of components.

Sample : Riboflavin (Vitamin B2)

Column       : Shodex AFpak AAF-894 (8.0mmID*50mm)
Eluent       : 0.1M 4-Ethylmorpholine acetate buffer(pH7.0)
Flow rate    : 1.0mL/min
Detector     : UV(260nm)
Column temp. : Room temp.

The components of vitamin B6 family resemble each other in structure and are difficult to separate. Usually, the method using ion pair reagent is used. However, under strong acidic conditions such as at pH 2.0, separation is possible by reversed phase mode using the slight difference of hydrophilicity. Three components of vitamin B6 family were separated with the Asahipak ODP-50 6D column, which can be used even at pH2.0.

Sample : 10micro-L
1. Pyridoxamine 2HCl 0.1mg/mL
2. Pyridoxal HCl 0.2mg/mL
3. Pyridoxine HCl (Vitamin B6) 0.12mg/mL

Column       : Shodex Asahipak ODP-50 6D (6.0mmID*150mm)
Eluent       : 50mM Sodium Phosphate buffer(pH2.0)
Flow rate    : 1.0mL/min
Detector     : UV(254nm)
Column temp. : 30deg-C

This shows an analysis example of vitamin B12 together with creatinine and urea by reversed phase mode using OHpak SB-802.5 HQ.

Sample :
1. Creatinine
2. Urea
3. Vitamin B12

Column       : Shodex OHpak SB-802.5 HQ (8.0mmID*300mm)
Eluent       : 10mM Phosphoric acid
Flow rate    : 1.0mL/min
Detector     : UV(210nm)
Column temp. : 40deg-C

RSpak KC-811 is suitable for the analysis of typical organic acids contain in fruits together with vitamin C.

Sample : 15micro-L
1. Citric acid 1350micro-g/mL
2. Malic acid 1020micro-gm/L
3. Vitamin C 610micro-g/mL
4. Succinic acid 1040micro-g/mL
Dissolved in 5% Methaphosphoric acid

Columns      : Shodex RSpak KC-811 (8.0mmID*300mm) x 2
Eluent       : 1mM HClO4 aq.
Flow rate    : 1.0mL/min
Detector     : UV(210nm)
Column temp. : 50deg-C

For the analysis of ascorbic acid, amino or silica based columns are usually used. Analysis using amino columns is particularly wide spread because isoascorbic acid as well as ascorbic acid can be analyzed together.
However, conventional amino columns are silica-based, bonded with aminopropyl groups or polyamine and are not chemically stable. Therefore, they have certain disadvantage such as a decrease in retention and short column life. Since Asahipak NH2P-50 4E is a polymer-based column, it does not suffer from such disadvantages. Moreover, since NH2P-50 column can be washed with alkaline solvents, the column life can be greatly extended.

Sample : (left) standards, (left) Grapefruit juice
1. D-Isoascorbic acid
2. L-Ascorbic acid

Column       : Shodex Asahipak NH2P-50 4E (4.6mmID*250mm)
Eluent       : 60mM Phosphoric acid(pH2.0)/CH3CN=20/80
Flow rate    : 1.0mL/min
Detector     : UV(254nm)
Column temp. : 40deg-C

Equilibration of NH2P-50 4E column : After passing 60mM Phosphoric acid in the column by 0.5 mL/min for 2 hours, it replaces by eluent.

For the analysis of ascorbic acid, amino columns or silica columns are usually used. Analysis using amino columns is particularly wide spread because isoascorbic acid (erythorbic acid) as well as ascorbic acid can be analyzed simultaneously. However, conventional silica-based amino columns are not chemically stable, therefore, they have certain disadvantage such as a decrease in retention and short column life. Since Asahipak NH2P-50 4E is a polymer-based column, it does not suffer from such disadvantages. Moreover, since NH2P-50 column can be washed with alkaline solvents, the column life can be extended.

Sample : 5micro-g/mL each, 10micro-L
1. Erythorbic acid
2. L-Ascorbic acid

Column       : Shodex Asahipak NH2P-50 4E (4.6mmID*250mm)
Eluent       : 20mM NaH2PO4+30mM H3PO4 aq./CH3CN=20/80
Flow rate    : 1.0mL/min
Detector     : UV(254nm)
Column temp. : 35deg-C

Equilibration of NH2P-50 4E column : After passing 60mM Phosphoric acid in the column by 0.5 mL/min for 2 hours, it replaces by eluent.

Vitamin D’s are ususally separated by normal phase mode or reversed phase mode. Vitamin D2 and D3 were separated using Asahipak ODP-50 6D ( polymer-based column ) by reversed phase mode, though they could not be separated using Silica 5SIL 4E ( silica-based column ) by normal phase mode. Since the usable pH range of the ODP-50 column is very wide and it can be washed with both acidic and alkaline solvents, it is very durable even if it is used for crude samples.

Sample : 4IU(0.1micro-g)/mL each, 100micro-L
1. Vitamin D2(Ergocalciferol)
2. Vitamin D3(Cholecaciferol)

Columns      : Shodex Asahipak ODP-50 6D (6.0mmID*150mm)  Shodex Silica 5SIL 4E (4.6mmID*250mm)
Eluent       : CH3CN/CH3OH=90/10                          Hexane/Isopropanol=99.6/0.4
Flow rate    : 1.5mL/min                                  1.6mL/min
Detector     : UV(265nm)
Column temp. : 27deg-C

Silica 5SIL 4D column is suitable for the separation of tocopherols. The chromatogram on the left shows the separation of alpha-, beta-, gamma- and delta-tocopherol by normal phase mode using the column with a mixture of n-hexane/isopropanol as an eluent. Asahipak ODP-50 6D is also suitable for the separation of tocopherols though alpha- and beta-tocopherol cannot be separated with the column. The chromatogram on the right shows the separation of alpha-, beta-, and gamma-tocopherol by reversed phase mode using the column with a methanol eluent.

Sample : Tocopherol
1. alpha-Tocopherol
2. beta-Tocopherol
3. ganna-Tocopherol
4. delta-Tocopherol

              (left)                                 (right)
Column       : Shodex Silica 5SIL 4D (4.6mmID*150mm)  Shodex Asahipak ODP-50 6D (6.0mmID*150mm)
Eluent       : n-Hexane/Isopropanol=99.5/0.5          CH3OH                     
Flow rate    : 1.0mL/min                              1.0mL/min
Detector     : UV(280nm)                              UV(295nm)
Column temp. : 30deg-C                                30deg-C

Carnitine (Vitamin BT) standard was separated using RSpak NN-814.

Sample : L-Carnitine (Vitamin BT) 0.1%, 20micro-L

Column       : Shodex RSpak NN-814 (8.0mmID*250mm)
Eluent       : 0.1M H3PO4 aq.  
Flow rate    : 1.0mL/min
Detector     : UV (210nm)
Column temp. : 25deg-C

For the analysis of vitamin B2, reversed phase columns are usually used with fluorescence detectors. This shows the analysis of vitamin B2 using Asahipak GS-320 7E (a multimode column).
GS-320 7E is an old type of GS-320 HQ.

Sample :
1. Flavin mononucleotide
2. Flavin adenine dinucleotide
3. 7alpha-Hydroxyriboflavin
4. Riboflavin (Vitamin B2)
5. Lumiflavin (LF)

Column       : Shodex Asahipak GS-320 7E (7.5mmID*250mm)
Eluent       : 1M Propionic acid buffer(pH4.4)
Flow rate    : 1.5mL/min
Detector     : Fluorescence (Ex. 250nm, Em. 520nm)
Column temp. : 55deg-C

For the analysis of vitamin B2, reversed phase columns are usually used with fluorescence detectors. This shows the analyses of vitamin B2 by reversed phase mode using Asahipak GS-320 7E, a multimode column.
GS-320 7E is an old type of GS-320 HQ.

Sample :
1. 7alpha-Hydroxyriboflavin
2. Riboflavin (Vitamin B2)

Column       : Shodex Asahipak GS-320 7E (7.5mmID*250mm)
Eluent       : 1M Propionic acid buffer(pH4.4)
Flow rate    : 1.5mL/min
Detector     : Fluorescence detector(Ex.250nm, Em.520nm)
Column temp. : 55deg-C

Nicotinic acid (niacin) in strawberry jam was analyzed using Asahipak NH2P-50 4E.

Sample : Pretreated strawberry jam
Niacin

Column       : Shodex Asahipak NH2P-50 4E (4.6mmID*300mm)
Eluent       : 75mM Sodium acetate/CH3CN=40/60
Flow rate    : 0.5mL/min
Detector     : UV(261nm)
Column temp. : Room temp.

S.Hirayama et al., J.Chromatgr., 588 (1991) 171

Vitamins in food were analyzed using RSpak DE-413 ( a column for reversed phase chromatography ).

Concentration Contents in 4g
1. Vitamin C
145.8micro-g/mL
87.5mg
2. Vitamin B6
21.2micro-g/mL
12.7mg
3. Vitamin B2
7.3micr-og/mL
4.4mg

 

Column       : Shodex RSpak DE-413 (4.6mmID*150mm)
Eluent       : (A); 1/30M Potasium phosphate buffer(pH6.8)/CH3CN=98/2
               (B); H2O/CH3CN=50/50
               gradient: 0min to 4min, 100% (A), 
                         4min to 15 min, 100% (A) to 30% (A), 
                         After 15 min, 30% (A) 
Flow rate    : 0.6mL/min
Detector     : UV(254nm)
Column temp. : 35deg-C

Nutrient beverage was analyzed using RSpak DE-413 ( a column for reversed phase chromatography ).

Concentration
Contents in 4g
1. Niacinamide
16.5micr-og/mL
24.7mg
2. Vitamin B1
9.4micro-g/mL
14.1mg
3. Vitamin B2
2.9micro-g/mL
4.3mg
 
Column       : Shodex RSpak DE-413 (4.6mmID*150mm)
Eluent       : (A); 1/30M Potasium phosphate buffer(pH6.8)/CH3CN=98/2
               (B); H2O/CH3CN=50/50
               gradient: 0min to 4min, 100% (A), 
                         4min to 15 min, 100% (A) to 30% (A), 
                         After 15 min, 30% (A) 
Flow rate    : 0.6mL/min
Detector     : UV(254nm)
Column temp. : 35deg-C

Vitamin C in soft drink was analyzed using Asahipak NH2P-50 4E.

(Sample pretreatment)
1. Add 5% methaphosphoric acid to make up 30mL sample solution.
2. Filtrate using a 0.45 micron disposable filter.
3. Just before the injection into HPLC, add purified water to make 10-fold solution.(The concentration of methaphosphoric acid should be 0.5% or lower.)

Sample : Soft drink containing Vitamin C

Concentration Contents in100mL
1. L-Ascorbic acid 37.2micro-g/mL 372mg 
Column       : Shodex Asahipak NH2P-50 4E (4.6mmID*250mm)
Eluent       : 20mM NaH2PO4+30mM H3PO4(pH2.2)/CH3CN=20/80
Flow rate    : 1.0mL/min
Detector     : UV(254nm)
Column temp. : 35deg-C

Equilibration of NH2P-50 4E column : After passing 60mM Phosphoric acid in the column by 0.5 mL/min for 2 hours, it replaces by eluent.

Vitamin A palmitate was analyzed using GPC KF-801. Wave length of 325nm at which it has maximum UV absorption was used for the detection. Usually, saponification is necessary to analyze vitamin A as the sample pretreatment. However, in this analysis, no complicated sample pretreatment procedure was necessary. What is necessary is just to dissolve the sample in the eluent(THF) and filtrate it using a 0.45 micron disposable filter. This method using KF-801 is a very simple way to analyze vitamin A.

Sample : Margarine 0.2%, 100micro-L
1. Vitamin A palmitate

Columns      : Shodex GPC KF-801 (8.0mmID*300mm) x 2
Eluent       : THF
Flow rate    : 1.0mL/min
Detector     : UV(325nm)
Column temp. : 40deg-C

Ascorbic acid and uric acid in blood were analyzed using Asahipak GS-320 7E ( a multimode column ) and an electrochemical detector (ECD) .
GS-320 7E is an old type of GS-320 HQ.

Sample :
Uric acid
Ascorbic acid

Column       : Shodex Asahipak GS-320 7E (7.5mmID*250mm)
Eluent       : 0.1M Sodium phosphate buffer(pH7.0) + 0.3M NaCl
Flow rate    : 1.0mL/min
Detector     : ECD(800mV VS Ag/AgCl)
Column temp. : 25deg-C

Courtesy of Dr. Iriyama, The Jikei University, School of Medicine

Ascorbic acid and uric acid in liquor cerebronspinalis were analyzed using Asahipak GS-320 7E ( a multimode column ) and an electrochemical detector (ECD).
GS-320 7E is an old type of GS-320 HQ.

Sample :
Uric acid
Ascorbic acid

Column       : Shodex Asahipak GS-320 7E (7.5mmID*250mm)
Eluent       : 0.1M Sodium phosphate buffer(pH7.0) + 0.3M NaCl
Flow rate    : 1.0mL/min
Detector     : ECD(800mV, VS, Ag/AgCl)
Column temp. : 25deg-C

Courtesy of Dr. Iriyama, The Jikei University, School of Medicine

Ascorbic acid in urine was analyzed using Asahipak GS-320 7E (a multimode column).
GS-320 7E is an old type of GS-320 HQ.

Sample : Urine
1. Glucose
2. Diketoglonic acid
3. Diketogluconic acid
4. Dehydroisoascorbic acid
5. Dehydroascorbic acid
6. Ascorbic acid
7. Isoascorbic acid

Column       : Shodex Asahipak GS-320 7E (7.5mmID*250mm)
Eluent       : The eluent was prepared by dissolving 4.5g of tartaric acid,
               1.5g of EDTA-2Na and 1g of beta-thiodiglycol in 2L of ultrapure water and 
               adjusting the pH to 3.0 with 4M sodium hydroxide solution.
Flow rate    : 1.0mL/min
Detector     : Fluorescence detector(Ex. 325nm, Em. 400nm)
               (Using post-column reaction)
Column temp. : 30deg-C

T.saki et al., J.Chromatogr., 385 (1987) 287.

Asahipak ODP-50 4E was used for the simultaneous analysis of fat-soluble vitamins. Good calibration curves were obtained for them.

Sample : 20micro-L
1. Vitamin K3 1.5micro-g/mL
2. Vitamin A 1.0IU/mL(0.3micro-g/mL)
3. Vitamin A acetate 0.5IU/mL(1.9micro-g/mL)
4. Vitamin D2 13.2IU/mL(0.3micro-g/mL)
5. Vitamin D3 13.2IU/mL(0.3micro-g/mL)
6. Vitamin E acetate 2.4micro-g/mL
7. Vitamin E 2.5micro-g/mL
8. Vitamin K1 2.4micro-g/mL

Column       : Shodex Asahipak ODP-50 4E (4.6mmID*250mm)
Eluent       : CH3CN/CH3OH=50/50
Flow rate    : 0.6mL/min
Detector     : UV(280nm)
Column temp. : 30deg-C

This shows the analysis of fat-soluble vitamins by GPC mode using GPC KF-801.

Sample : 100micro-L
1. Vitamin A palmitate 25IU/mL(500micro-g/mL)
2. Vitamin E 50micro-g/mL
3. Vitamin D3 50micro-g/mL
4. Vitamin K3 50micro-g/mL


Columns      : Shodex GPC KF-801 (8.0mmID*300mm) x 2
Eluent       : THF
Flow rate    : 1.0mL/min
Detector     : UV(280nm)
Column temp. : 40deg-C

Naturally occurring Vitamin E is classified into two categories: tocopherols and tocotrienlols, each containing 4 subcategories, and therefore amounting to a total of 8 homologs. Tocopherols exhibit a higher bioactivity, the most active type being α-tocopherol, and for this reason the term vitamin E generally refers to α-tocopherol. In this application, the silica 5SIL 4D normal phase column was used to remove β-tocophenol and analyze simultaneously 7 kinds of tocopheros and tocotrienols.

Sample : Vitamin E, 20micro-L
1. α-Tocopherol 5μg/mL
2. α-Tocotrienol 10μg/mL
3. β-Tocopherol 5μg/mL
4. γ-Tocopherol 5μg/mL
5. γ-Tocotrienol 10μg/mL
6. δ-Tocopherol 5μg/mL
7. δ-Tocotrienol 10μg/mL

Column       : Shodex Silica 5SIL 4D (4.6mmID*150mm)
Eluent       : n-Hexane/Isopropanol/CH3COOH=1000/6/5                   
Flow rate    : 1.0mL/min
Detector     : Fluorescence(Ex. 298nm, Em. 325nm)
Column temp. : 30deg-C