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 4.6mm
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
FixedDual
WavelengthDetector
Photodiode
ArrayDetector
•
16
•
800-356-1688 •
2005vol. 3
Sudandyes are synthetic industrial azo-dyes traditionallyused inwaxes, plas-
tics, oils, and polishes. Although recognized as carcinogens, Sudan dyes
recentlyhavebeen found in foodproducts in someEuropean countries.They
are added to foods such as chili powders tomimic, intensify, andprolong the
appearance of natural red hues. In theUK,more than six hundred products
containingSudandyeshavebeen recalled, the largest food recall inBritishhis-
tory.¹
Sudandyes are categorizedasClass 3 carcinogensby the InternationalAgency
for Research on Cancer (IARC) and, therefore, are illegal as food additives
according to both the FDA and the EU. The EuropeanCommission requires
products to have documentation confirming the absence of Sudan dyes.²,³
Since2003,Europeannationshave required randomproduct testingand test-
ing of suspected adulterated products. Items found to contain Sudan dyes
must be disposedof as hazardouswaste.
4
Laboratories performing analyses for Sudan dyes are not required to follow
definedmethods.TheEUhas setdetection limitsat0.5-1mg/kg,andany food
material containingmore than the limit shouldbewithdrawn from themar-
ket.¹Here, we describe a simple reversedphaseHPLC 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 inHPLC 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 dilutingwithmethanol to 20µg/mL each dye.We used a
150x4.6mmUltraAqueousC18HPLCcolumn (cat.#9178565) for theanaly-
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 493nmor 604nm, Sudan III at 508nm to512nm, andSudan IV at
357nm or 520nm. For each dye except Sudan III, we observed the higher
response at the first listedwavelength; for Sudan III therewas littledifference.
The dyes can be detected bymonitoring at 488nm for Sudan I and II and at
520nm for Sudan III and IV, allowing all four dyes tobedetectedwith a fixed
dual wavelength instrument.
Thismethod is simple, yet efficient, requiringonlya simplemobilephase, iso-
cratic elution, anddetection at twowavelengths. TheUltraAqueousC18 col-
umnprovides the selectivityneeded to assure the separation.
SimpleHPLCAnalysis forSudanDyes
Monitor Sudan I, II, III, and IV inaSingle, IsocraticAnalysis
By Julie Kowalski, Innovations Chemist
• UltraAqueousC18HPLC column separates the four Sudandyes in20minutes.
• Simplemethanol andwatermobilephase; twowavelengthsdetect all four dyes.
• Twowavelengthsdetect all four dyes.
for
more
info
For other column dimensions, please refer to our catalog, or visit ourwebsite.
References
1.
2. CommissionDecision of 20 June 2003 on emergencymeasures 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 CommissionDecision 2003/460/EC of 21 January 2004.
4.
UltraAqueousC18Column (USPL1)
5µmColumn, 4.6mm
cat.#
150mm
9178565
Figure1
Monitor Sudan I, II, III and
Sudan IV ina single, isocratic analysis.
