Food colors separation Tartarzine & Allora red by HPLC

Discussions about HPLC, CE, TLC, SFC, and other "liquid phase" separation techniques.

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Hellow every

firs thanks so much for every one he try to help me . in my laboratory we have HPLC perkin elmer company . and i want separate food artificial colors ( tartrazine and allorared ) in juices and beverages . i tried but i got retention time early and poor resolution ( 1.91 for tartrazine standard and 2.32 for allora red standard ) -Column is C 18 , 250 mm , 4.6 mm . mobile phase was ( 10 % ACN : 90 % OPA 0.1 % ) . OPA = Orthophosforic Acid . and flow rate = 1.0 ml \ mint and run time 10 mint .
so please if any one can help me for this experiment ?

THANK YOU FRIENDS .
The truth is out there!
Actually, just do a Google search——there are several HPLC methods for either tartrazine or allura red; just modify for your purpose.
If you wish to use your current method, single easiest modification is to lower flow rate to say 0.5 mL/ min or even less.
Google search is a good idea. Reducing the flow rate not so much.

A 250 x 4.6 mm column has an internal volume ("dead volume") somewhere around 2.5 mL, so at 1 mL/min will have a dead time somewhere around 2.5 minutes. That means that both peaks are very weakly retained by (if not actually excluded from) the stationary phase.

10% ACN / 90% buffer is an extremely weak mobile phase, which makes me suspect that either your column is dead or you are actually looking at system peaks ("t0 noise") rather than your actual analytes. If you have access to a diode array detector it might be a good idea to scan the absorbance spectrum of one or both peaks to confirm that they match those of the analytes. If they do, it's probably a dead column. If they don't, they are probably system peaks and your actual analytes are stuck on the column (try running a gradient and see if anything elutes).
-- Tom Jupille
LC Resources / Separation Science Associates
tjupille@lcresources.com
+ 1 (925) 297-5374
Thank you so much ( JMB & tom jupille ) for your response .


Mr : JMB :

i will first reduce flow rate to 0.5 ml \ mint for the same mobile phase . and see retention time ? . and the resolution .

-------------------------------------------
Mr : tom jupille :

do you have a gradient program for this mobile phase ? how can i start .how can make this mobile phase more strong ? Any suggestions ؟
my columns is new & my HPLC detector is DAD detector .


Thank you again for help .
I am finishing several methods for a company for analysis of food dyes in beverages, drugs, etc. For tartarzine and Allora red we used Amaze TH HILIC/ion-exchange column (3.2x50 and 3.2x100 mm). There is absolutely no issue with retention and you can get K'>3 and use 50 mm column. I will apply this method for ssome Coke or Gatorade and can share results with you later this week. Please send me an email through our website if you are interested.
Vlad Orlovsky
HELIX Chromatography
My opinions might be bias, but I have about 1000 examples to support them. Check our website for new science and applications
www.helixchrom.com
do you have a gradient program for this mobile phase ? how can i start .how can make this mobile phase more strong ?
The short answer is "add more acetonitrile". That said, I get the impression that you are new to HPLC. You might benefit from the "Fundamentals of HPLC" course provided by our hosts.

Since you have a DAD, have you collected spectra on your peaks to verify that they actually match the analytes?
-- Tom Jupille
LC Resources / Separation Science Associates
tjupille@lcresources.com
+ 1 (925) 297-5374
Mr : Vlad Orlovsky .

Tank you for your reply . a bout the columns i do not have HILIC column . only C18 column ( 250 mm , 4.6 mm , 5 um ) . if you have any methods we can do it and send you what i get results .


thank you again ( Vlad Orlovsky ) .


my email : ( khaled_ma_2000@yahooo.com ) .
If you want to use regular C18 column, then you can:
- use low organic (5%) in combination with low pH (sulfuric or TFA in the mobile phase)
- use ion-pairing reagent

You can also use IE or mixed-mode column.
Vlad Orlovsky
HELIX Chromatography
My opinions might be bias, but I have about 1000 examples to support them. Check our website for new science and applications
www.helixchrom.com
Mr : Vlad Orlovsky .

thank you for all your response . but can you tell me clearly what is the separate conditions can i follow ? i mean mobile phase composition and flow rate and so on ?

thank you Mr : Vlad Orlovsky
Do the following for the P column you have:
- prepare standard of your dye (0.1-0.2 mg/ml) in ACN/water (10/90)
- wash column for 30 min with 10% ACN and 0.1% sulfuric acid
- inject standard without column and measure peak area
- inject standard with the column and measure peak areas of all peaks. The sum of all peaks should match peak area of the single peak without the column.

If retention is too short you can try to reduce ACN to 5%, if it is too long, increase amount of ACN to 20%.

If you have no retention, then use basic ion-pairing reagent like tetrabutylammonium (10-30 mM)
Vlad Orlovsky
HELIX Chromatography
My opinions might be bias, but I have about 1000 examples to support them. Check our website for new science and applications
www.helixchrom.com
here are methods for analysis of tartrazine, Sunset Yellow, and Allura Red (Yellow 5, Yellow 6 and Red 40 dyes)

http://helixchrom.com/applications/hplc ... de-column/

http://helixchrom.com/applications/hplc ... de-column/

This method can be used to analyze all acidic dyes.
Vlad Orlovsky
HELIX Chromatography
My opinions might be bias, but I have about 1000 examples to support them. Check our website for new science and applications
www.helixchrom.com
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