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INTRODUCTION

Synthetic dyes are used to improve the visual

appeal of many of the products we use every

day. Bright, eye-catching colors in foods,

cosmetics, pharmaceuticals, and household

cleaners help to promote sales, but generally do

not improve quality.1 Although several dyes are

currently approved for market, many are

associated with a variety of adverse side effects

including behavior problems in children, cancer,

and allergic reactions.2,3 The structural variation

of the starting materials used to synthesize

dyes creates complex mixtures of product

containing isomers and subsidiary colors. It is,

therefore, imperative to efficiently isolate

impurities for subsequent identification and

characterization. The constituents in a

synthetic dye mixture are structurally similar,

leading to challenges in chromatographic

resolution. Furthermore, the typically low levels

of the impurities with respect to the main

compound complicate their isolation. In this

study, we illustrate the advantages of mass-

directed purification for the isolation of isomers

as well as other impurities from commercially

available FD&C Blue 1. Scaleup from analytical

to preparative separation, sample introduction

techniques, and the impact of MS detection on

the overall purification efficiency will be

discussed.

IMPROVING ISOLATION EFFICIENCY OF IMPURITIES IN SYNTHETIC DYES USING MASS-DIRECTED PURIFICATION

Jo-Ann M. Jablonski*, Andrew J. Aubin, and Thomas E. Wheat

Waters Corporation, 34 Maple Street, Milford, MA 01757

*Corresponding author: jo-ann_jablonski@waters.com

References

1. http://www.forbes.com/sites/rachelhennesset/2012/08/27/living-in-color-the-potential-dangers-of-artificial-dyes

2. T. Zou, P. He, A. Yasen, Z. Li, “Determination of seven synthetic dyes in animal feeds and meat by high performance liquid chromatography with diode array and tandem mass detectors”, Food Chemistry, 138 (2013) 1742-1748

3. D. Wallinga, M.D., Institute for Agriculture and Trade Policy, http://www.iatp.org/documents/smart-guide-to-food-dyes-buying-foods-that-can-help-learning, 2009

4. Jablonski JM, Wheat TE, Diehl DM. Developing Focused Gradients for Isolation and Purification. Waters Application Note 720002955EN. 2009 September.

5. Wheat TE, et.al. At-Column Dilution Application Notes. Waters Application Note 71500078010rA. 2003

Conditions

Analytical Column: XBridge® C18, 4.6x50 mm, 5 µm Analytical flow rate: 1.46 mL/min

Prep Column: XBridge C18 OBD™ Prep, 19x50 mm, 5 µm Preparative flow rate: 25 mL/min

Mobile phase A: 0.1% ammonium hydroxide in water Mobile phase B: Acetonitrile, neat

Makeup solvent: 90% water/10% acetonitrile, 0.01% ammonium hydroxide

Wavelength: 628 nm Gradients and injection volumes: as noted in figures

Sample: FD&C Blue 1, 10 mg/mL in water Instrumentation

Waters AutoPurification™ System: 2545 Binary Gradient Module, 2767 Sample Manager, System Fluidics Manager, 8-30

Flow Splitter; two 515 HPLC pumps, 2998 Photodiode Array

Detector, ACQUITY® QDa™ Detector

CONCLUSION

EXPERIMENTAL

RESULTS AND DISCUSSION

Figure 1. Structure and total ion chromatograms for FD&C Blue

1; monoisotopic mass 792.1, doubly-charged ion mass 373.3. Analytical gradient: 5-50%B in 5 min. Injection volume: 5 µL.

Figure 2. Overlay of positive and negative ion mode single ion

recording (SIR) chromatograms for the analytical and focused gradients. Injection volumes: 5 µL.

Focusing the gradient4 increases the resolution between

compounds and provides a greater opportunity for increased column loading, thereby reducing the number of runs required

to isolate the product, as shown in Figure 2.

A loading study performed using the new focused gradient on

the analytical column resulted in a determination that the maximum injection volume of 50 µL maintained the resolution

between component peaks . As shown in Figure 3, geometric scaling to the 19x50 preparative column demonstrated good

separation and collection of the two target peaks.

Figure 3. SIR of 853 µL (8.5 mg) prep injection with collection

of the product and impurity peaks.

Figure 4. A tee placed at the head of the column dilutes the

sample with initial mobile phase composition, concentrating the sample at the head of the column until the gradient begins.

Figure 5. SIR of 2.6 mL (25.6 mg) At-Column Dilution prep in-

jection with collection of the product and impurity peaks. Even with 3x increase in sample load, peak resolution is equivalent

to or slightly better than the geometrically scaled conventional injection shown in Figure 3.

Fraction analysis (not shown) revealed excellent purity for the

product collections (Fr 4 and 18, 100%). The isomer/impurity (Fr 23 , 24) were 77% and 100%, respectively. The positive

ion impurity (Fr 13, 31) were 94% and 98% pure, respectively.

Mass-directed purification efficiently targets product as well as impurities from complex sample mixtures,

reduces the number of collected fractions, and when used with focused gradients and at-column dilution,

saves time, and increases sample throughput. Mass detection in both positive and negative modes

improves the probability of finding additional impurities and leads to higher product purity.

Focusing the gradient improves the resolution between compounds and leads to higher column

loading, reducing isolation time. At-Column Dilution increases the column mass

capacity for better isolation efficiency.

Using a generic 5-50% gradient provides a separation of the

components of interest, as shown in Figure 1. However, in order to isolate the quantities of sample required, further

improvements to the method were required.

Isolation efficiency improves with higher column loading,

reducing the number of repetitive chromatographic runs required to obtain sufficient product for ensuing experiments.

As shown in figures 4 and 5, sample introduction using the At-Column Dilution5 technique increased the prep column loading

three-fold.

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