amu atomic mass units
approx. approximately
FA formic acid
Fig. Figure h hours
HPLC high pressure liquid chromatography M molar
min minute mL millilitre
N number of observations
na not applicable
nc not calculable
No. number
ns no sample
nsr no sample for re-analysis sec seconds Tab. Table
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3. Table of contents
1. Synopsis ... 2
2. List of abbreviations and terms... 3
3. Table of contents... 4
12. Figures not included in the Text... 16
12.1. Figures ... 16
12.1.1. Chromatograms ... 16
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4. Catalogue of tables and figures
Tab. 1: Peak values (main peak) of different dyes in acetone: olive oil (1+3) at different time
points... 13
Fig. 1: Example chromatogram of dye A ... 16
Fig. 2: Example chromatogram of dye B... 17
Fig. 3: Example chromatogram of dye C... 17
Fig. 4: Example chromatogram of dye D ... 18
Fig. 5: Example chromatogram of dye E... 18
Fig. 6: Example chromatogram of dye F ... 19
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5. Signatures
Munich, date Franziska Anzenberger
study performance
Munich, date Dr. Michael Schulz
study director
Munich, date Ingrid Lamprecht
study report
Munich, date Dr. Alexander von Nieciecki
head test facility
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6. Introduction
The objective of this study was testing the stability of 6 different dyes in a mixture of acetone and olive oil (1+3) as vehicle.
The dyes were suspended in the vehicle in concentrations of 15 % /dye A – E) and 9 % (dye F). The dyes were not soluble in these concentrations in this vehicle.
The following dyes were used:
BSL Nummer Test item 053226A YELLOW E-JD 3442 053226B C.I. Reactive Red 231 053226C Produkt P-4G
053226D C.I. Reactive Yellow 174 053226E NAVY 14 08 723
053226F Dispersionsrot 2754
To simplify matters the dyes were furthermore named A – F.
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7. Summary
The dyes were suspended in an acetone/olive oil mixture, so it was difficult to take
homogeneous samples, due to the distribution of different sized dye particles. Therefore some differences in peak size did not result from stability but of difficult sample handling.
Dye B showed no degradation in between 5 hours, also dye F which gave two peaks. Only one peak was selected for calculation. Different peak areas were found for Dyes A and C which are resulting of the difficult handling of the suspension, but also no degradation was observed. The peak areas of Dye D and E were stable if the areas of the two main peaks were added.
8. Principle
8.1. Method description
The pigments were weighed into a PP-tube in a concentration according to the following table:
1 ml of the vehicle was pipetted to the dyes and mixed on a vortex mixer. 10 µL were withdrawn at following time points: 0 h, 0.5 h, 1 h, 2 h and 5 h and then diluted with methanol.
Due to the insolubility of the dyes in the vehicle the dilution was very problematic. The different peak values could be explained thereby.
Dye D and E the chromatograms were not reproducible the first time, so they were weighed five-fold (for each time point separately) the half amount and mixed with 0.5 mL of vehicle.
Subsequently 0.5 mL water were added and mixed. After a holding time of 15 minutes the upper phase was suspended and an aliquot of the residue was diluted with water and injected.
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9. Experimental part
Instrumentation and reagents Chemicals
Water (prepared by Milli-Q plus) Millipore, 65760 Eschborn, Germany
Methanol Merck KGaA, 64271 Darmstadt,
Germany
Aceton No. 34850 Sigma-Aldrich, 91625 Schnelldorf,
Germany
AV059321 Alltech Associates, Inc; 82008 Unterhaching, Germany 0.3 mL PP-short thread micro-vial
32x11.6 mm
AV059321 Alltech Associates, Inc; 82008 Unterhaching, Germany
5 mL tubes with caps, 57x15.3 mm Sarstedt, 51588 Nümbrecht, Germany General laboratory equipment and glassware
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Laboratory equipment
Half micro balance AT261, DeltaRange, Mettler, 35396 Gießen, Germany Autom. Pipettes, various volumina Eppendorf Reference, 22331 Hamburg, Germany
Multipette pro, Eppendorf, 22331 Hamburg, Germany
Shaker Jahnke und Kunkel KG,
IKA-Vibrax-VXR, 79219 Staufen i. Breisgau,
Auto sampler (cooled to 5 ºC)
Alliance, Waters 2695 Separations Module, Waters, 65760 Eschborn, Germany
Analytical column ReproSil Pur ODS 3 5µ 150x4.6mm, Maisch , 72119 Ammerbuch 3, Germany
Data system (quantitative evaluation)
Millennium32 (Version 3.20), Waters, 65760 Eschborn, Germany
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Modell Photodiode Array Detector, Waters 996
Detection wavelength 234 nm
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10. Results
The peaks of each chromatogram were integrated and the peak areas of the main peak at different time points were compared. For none of the dyes a constant loss of peak area was indicating degradation. Especially in the case of dye A large variations were observed, but with no clear tendency. These variations were due to problems in the sampling from
suspension, possibly caused by large variations in the size clots aggregates) formed by the dye particles in this oily suspension.
In addition each chromatogram was checked for peaks of potential degradation products. In none of the chromatograms any additional peaks were raising during incubation time. This confirms the conclusion that no degradation of any of the dyes under investigation is occurring.
Tab. 1: Peak values (main peak) of different dyes in acetone: olive oil (1+3) at different time points
Dye A (repeat measurement) Dye B
Time points Peak area
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Dye D Dye E
Time points
Peak area D1+D2
%
Time points
Peak area E1 + E2
%
start 92.7 start 100.0
0.5 h 97.8 0.5 h 100.0
1 h 98.1 1 h 100.0
2 h 98.2 2 h 100.0
5 h 91.7 5 h 100.0
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11. Archiving
After completion of the study all chromatographic raw data (chromatograms, methods, summary tables), MS Excel tables and MS Word documents (study protocol, report) were saved on the BioProof network. The following documents are archived in the BioProof archive:
• laboratory notebooks and sample tables
• raw data and interim results as computer print-outs (calibration lines, chromatograms, reports, excel tables)
• report
• CD-ROM comprising all relevant data
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12. Figures not included in the Text
12.1. Figures
12.1.1. Chromatograms
Fig. 1: Example chromatogram of dye A
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Fig. 2: Example chromatogram of dye B
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Fig. 4: Example chromatogram of dye D
Fig. 5: Example chromatogram of dye E
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Fig. 6: Example chromatogram of dye F