Arkansas Department of Health Division of Public Health Laboratories

1
Arkansas Department of HealthDivision of Public
Health Laboratories
Simultaneous Detection of Color Additives in Food
and Beverage Products by HPLC

• Sandra Washko, Food Chemistry
• Lab Supervisor

2
Introduction

• Regulations
• 1886 - color added to butter
• 1906 - Pure Food and Drugs Act administered by
  USDAs Bureau of Chemistry
• 1938 - Food Drug and Cosmetic Act, use of
  certified colors became mandatory
• 1960 - U.S. Color Additives Amendment

3
Introduction Cont.

• Permitted Categories
• Natural exempt from certification plant,
  animal, mineral
• Synthetic subject to certification coal tar
  sources

4
Terminology

• Colorant, dye, lake, pigment, natural, synthetic,
  water-soluble, oil-soluble, common name, numbers,
  chemical class, certified, provisional,
  inorganic, stability, extract.
• CAS number. Established by the American Chemical
  Society.
• CI number. International numbering system
  established by the Society of Dyers and Colorists.

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Terminology Cont.

• E number. Established by the European
  Community.
• FDC. Official nomenclature in USA for certified
  colorants.
• Sunset Yellow FCF FDC Yellow 6, E110, CI Food
  Yellow 3, CI 15985, CAS 2783-94-0, chemical
  name disodium salt of 1-p-sulphophenylazo-2-napht
  hol-6-sulfonic acid, chemical class azo.

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Certified Dye Standards
FDC No. Absorbance, nm
Selected Wavelength Yellow
5 428 450 Yellow 6 484
Red 40 507 520 Red
3 525 Blue 2 610 620 Green
3 625 Blue 1 630
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Principle of Method

• Water-soluble compounds
• Separated on reversed phase C18
• Colorant is ionized-polar compound
• Adjust to pH 5 to retain non-polar molecules on
  C18 column
• Apply alcohol to polarize and elute compounds
• Detect absorption in visible spectrum

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Instrumentation

• HPLC, quaternary pump, programmable-wavelength
  detector.
• Temperature 30C
• Flow 1.5 mL/min.
• Inject 20 uL
• Run Time - 35 minutes

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HPLC
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Instrumentation Cont.

• Mobile Phase
• A - 0.005M Ammonium Acetate, pH 5
• B - Methanol
• C - Water
• Gradient Programs
• Condition - 100 methanol to 100 water

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Instrumentation Cont.

• Analysis - Buffer Methanol Time
• 100 0 0
• 60 40 10
• 30 70 25
• 100 0 35
• Shut-down - 100 buffer to 100 water to
  100 methanol

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Standard Mixture

• Weigh one mg of each dye.
• Bring to 100 mL volume with 10 buffer in water

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Sample Preparation

• Solid Let sit in 10 mL buffer.
• Beverage Dilute if color is intense.
• C18 SPE Column condition - 2 mL methanol, dry
  2 mL water, dry.
• Apply all of sample filtrate.
• Apply 2 mL standard dye mixture.
• Wash column with 1 mL buffer.
• Extract the dye(s) with 2 mL methanol.
• Filter through Acrodis syringe filter to an AS
  vial.

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Standard applied to SPE column.
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Standard retained on SPE column.
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Standard eluted from SPE column.
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Yellow Spectrum
Yellow 6
Yellow 5
Red 40
mV
450 nm
0
5
15
20
25
30
35
10
Minutes
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Red Spectrum
Yellow 6
Red 3
Red 40
mV
520 nm
0
5
15
20
25
30
35
10
Minutes
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Blue Spectrum
Blue 1
Green 3
Blue 2
mV
620 nm
0
5
15
20
25
30
35
10
Minutes
20
RainbowSpectra
Blue 1
Green 3
Blue 2
Yellow 6
Red 3
Yellow 5
Red 40
mV
620 nm
520 nm
450 nm
0
5
15
20
25
30
35
10
Minutes
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Laboratory Sample
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Ingredient List
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PearSample
Yellow 6
Yellow 5
mV
620 nm
520 nm
450 nm
0
5
15
20
25
30
35
10
Minutes
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Conclusion

• Rapid screen for variety of samples
• One injection for all certified colors
• Expense - minimal after purchase of HPLC
• Easily fine-tuned for unknowns
• MDL is 1.25 ppm

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References

• Anonymous. Code of Federal Regulations, 21 CFR
  70-82. U.S. Government Printing Office,
  Washington, DC, 1998.
• Bell, Sandra, ed.Official Methods of Analysis of
  AOAC International, 16th ed, 4th Revision, Vol.
  II, Chap. 46, Gaithersburg, Maryland, 1998.
• Anonymous. Warner-Jenkinson brochure, Certified
  Food Colors. 1998.
• Tanner, James T, Food Testing and Analysis, 4(6)
  19-20 (1999).
• Frick, Dave and Penny Huck, Cereal Foods World,
  40(4) 209-218 (1995).

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Sandra WashkoArkansas Department of Health4815
West Markham, Slot 47Little Rock, AR
72205-3867Phone [phone]Fax
501-661-2468email swashko_at_mail.doh.state.ar.us