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Sample:
Inj.: 20µL, mixture of Sudan I,
Sudan II, Sudan III, Sudan IV
Conc.: 20µg/mL each component
Sample diluent: methanol
Column: Ultra Aqueous C18 (cat.# 9178565)
Dimensions: 150 x 4.6 mm
Particle size: 5µm
Pore size: 100Å
Conditions:
Mobile phase: methanol:water, 97:3
Flow: 1mL/min.
Temp.: ambient
Det.: UV @ 476 / 493 / 512 / 357 nm (top) or
UV @ 488 / 520 nm (bottom)
1. Sudan I (476nm)
2. Sudan II (493nm)
3. Sudan III (512nm)
4. Sudan IV (357nm)
1. Sudan I (488nm)
2. Sudan II (488nm)
3. Sudan III (520nm)
4. Sudan IV (520nm)
LC_FF0326
LC_FF0327
Fixed Dual
Wavelength Detector
Photodiode
Array Detector
• 16 • 800-356-1688 • 2005 vol. 3
Sudan dyes are synthetic industrial azo-dyes traditionally used in waxes, plas-
tics, oils, and polishes. Although recognized as carcinogens, Sudan dyes
recently have been found in food products in some European countries. They
are added to foods such as chili powders to mimic, intensify, and prolong the
appearance of natural red hues. In the UK, more than six hundred products
containing Sudan dyes have been recalled, the largest food recall in British his-
tory.¹
Sudan dyes are categorized as Class 3 carcinogens by the International Agency
for Research on Cancer (IARC) and, therefore, are illegal as food additives
according to both the FDA and the EU. The European Commission requires
products to have documentation confirming the absence of Sudan dyes.²,³
Since 2003, European nations have required random product testing and test-
ing of suspected adulterated products. Items found to contain Sudan dyes
must be disposed of as hazardous waste.4
Laboratories performing analyses for Sudan dyes are not required to follow
defined methods. The EU has set detection limits at 0.5-1 mg/kg, and any food
material containing more than the limit should be withdrawn from the mar-
ket.¹ Here, we describe a simple reversed phase HPLC separation of Sudan I,
Sudan II, Sudan III, and Sudan IV (Scarlet Red).
We prepared 1mg/mL stock solutions of Sudan I or Sudan II in HPLC grade
methanol, and equivalent solutions of Sudan III or Sudan IV in ethyl acetate.
To avoid reductive cleavage, we stored the stock solutions at 4°C in foil-
wrapped containers. We prepared sample solutions by combining the four
stock solutions and diluting with methanol to 20µg/mL each dye. We used a
150 x 4.6mmUltra Aqueous C18 HPLC column (cat.# 9178565) for the analy-
sis.
Results
Figure 1 shows the Ultra Aqueous C18 column separates the four dyes in
approximately 20 minutes. Sudan I can be detected at 476nm or 418nm,
Sudan II at 493nm or 604nm, Sudan III at 508nm to 512nm, and Sudan IV at
357nm or 520nm. For each dye except Sudan III, we observed the higher
response at the first listed wavelength; for Sudan III there was little difference.
The dyes can be detected by monitoring at 488nm for Sudan I and II and at
520nm for Sudan III and IV, allowing all four dyes to be detected with a fixed
dual wavelength instrument.
This method is simple, yet efficient, requiring only a simple mobile phase, iso-
cratic elution, and detection at two wavelengths. The Ultra Aqueous C18 col-
umn provides the selectivity needed to assure the separation.
Simple HPLC Analysis for Sudan Dyes
Monitor Sudan I, II, III, and IV in a Single, Isocratic Analysis
By Julie Kowalski, Innovations Chemist
• Ultra Aqueous C18 HPLC column separates the four Sudan dyes in 20 minutes.
• Simple methanol and water mobile phase; two wavelengths detect all four dyes.
• Two wavelengths detect all four dyes.
for more info
For other column dimensions, please refer to our catalog, or visit our website.
References
1.
2. Commission Decision of 20 June 2003 on emergency measures regarding hot chili and hot chili products,
notified under document number C(2003) 1970, (2003/460/EC), OJ L. 154/114, 21.6.2003.
3. Implementation of Commission Decision 2003/460/EC of 21 January 2004.
4.
Ultra Aqueous C18 Column (USP L1)
5µm Column, 4.6mm cat. #
150mm 9178565
Figure 1 Monitor Sudan I, II, III and
Sudan IV in a single, isocratic analysis.