manual of methods for determining micronutrients in fortified foods

• Beakers (25, 100 and 1000 mL)
• Glass funnels
• Amber glass vessels
• Volumetric pipettes
• Graduated cylinders
• Centrifuge tubes (50 and 10 mL)
• Test tubes (10 mL)
• Glass rods
• Filter paper Whatman No. 41
V. Reagents
1. Acetic acid - 0.02M: ((CH 3
COOH), 99.8%, FW: 60.05, d=1.05 g/mL). In a 1L volumetric flask
containing around 500 mL of distilled water add 1.2 mL of glacial acetic acid. Agitate and make up
to volume with distilled water.
2. Amylase-5% w/v: Weigh 1.25 g amylase and add around 5 mL distilled water. Let it stand until
is completely hydrated. Make up to volume 25 mL with distilled water and agitate thoroughly.
Prepare only the amount to be used.
3. Sodium acetate-2M: (CH 3
COONa), 99.5%. Dissolve 164 g of sodium acetate anhydrous in
distilled water and dilute to 1 L.
4. Sulfuric acid-0.1M: (H 2
SO 4
, 95-97%, 1.84 g/mL, FW 98.08). In a beaker containing around 600 mL
distilled water, add 10 mL concentrated sulfuric acid. Agitate and make up to 1 L with distilled water.
5. Mobile phase (HTAA:Methanol, 83:17)
9
Solution A, HTAA (Sodium hexanosulfonate-5mM, triethylamine-0.13 %, acetic acid-1%)
In a 25-mL beaker weigh 0.9602 g sodium hexanosulfonate (C 6
H 13
O 3
SNa, Sigma Ultra 98%, FW:
188.2 g/mol, Sigma H-9026). Dissolve in HPLC grade water and transfer quantitatively to a 1-L
volumetric flask. Add 1.3 mL triethylamine ((C 6
H 15
N), > 99%, FW 101.19, d=0.73 g/mL) and 10
mL glacial acetic acid ((CH 3
COOH), 99.8%, FW=60.05, d=1.05 g/mL). Make up to volume with
HPLC grade water.
In a 1-L Erlenmeyer flask add 830 mL solution A and 170 mL methanol. Mix well and filter the
solution through a 0.45 µm filter. Degas the solution prior to use. Prepare only the volume of
mobile phase to be used.
9 Dong, M., Lepore, J. y Tarumoto, T. 1988. Factors Affecting the Ion-Pair Chromatography of Water Soluble Vitamins. J. Chromatogr.
442: 81-95.
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