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2007 vol. 2

Foods, Flavors & Fragrances

Simple, Reliable HPLC Analyses of Organic Acids

Using Water-Compatible Allure® or Ultra C18 Columns

Julie Kowalski, Ph.D., Innovations Chemist, and Becky Wittrig, Ph.D., HPLC Product Marketing Manager

• Use 100% aqueous mobile phases without losing retention.

• Simple, isocratic method.

• Complete resolution of critical fruit juice organic acids, including quinic and tartaric acids.

Organic acids are common components in foods and beverages, and play a critical role in product characteristics like taste and

aroma. They can be tested for in many food products including fruits, cheeses, and various beverages such as juices and wines.

Organic acids can originate in the foods themselves (e.g. cranberries) or can be produced by food processing (e.g. alcoholic fermentation).

A method that allows resolution of organic acids, as well as their quantification, can help determine product quality and authenticity.

Reversed phase HPLC coupled with UV-Vis detection is a popular technique for organic acid analysis. One common method, AOAC

method 986.13, stipulates reversed phase HPLC using two C18 stationary phase columns in series. Because organic acids are low in

molecular weight, and have polar functionalities, 100% aqueous buffer is needed for adequate retention. A low pH buffer is used to

ensure that the organic acids remain protonated or neutral, thus allowing the best interaction between the organic acids and the C18

stationary phase. However, using a 100% aqueous mobile phase can cause the C18 chain in conventional C18 columns to collapse.

Phase collapse results in loss of retention, and the column must be flushed with organic mobile phase, a time consuming step, to

restore chain structure and column performance.

Three Restek columns – the Ultra Aqueous C18 column, the Allure® Aqueous C18 column, and the Allure® Organic Acids column

– use aqueous-compatible C18 phases that do not exhibit phase collapse, even with 100% aqueous mobile phases. The advantage of

using these columns is demonstrated in Figure 1 by the fast analysis of organic acids on a Shimadzu Prominence 20A system. Here,

we compared the ability of the Ultra Aqueous C18 phase and a conventional C18 phase to withstand phase collapse. Figures 1A and

1B show that the Ultra Aqueous C18 phase resolves organic acids in a 100% aqueous mobile phase without loss of retention. In com-

parison, the conventional C18 phase shown in Figure 1C and 1D suffers a complete loss of retention following phase collapse when

used under the same conditions. Thus, in an analysis that requires, or is improved by, a mobile phase with a high aqueous content,

an Ultra Aqueous C18 column is the superior choice.

Figure 1

Restek’s water-compatible C18 phase does not collapse in a 100% aqueous mobile phase, compared to a

conventional C18 column which shows a complete loss of retention.

Instrument: Shimadzu Prominence 20A

Sample: Inj.: 10μL; Conc.: 2000μg/mL each component except fumaric acid (10μg/mL) (Organic Acids Reference Mixture cat.# 35080); Sample diluent: deionized water

Conditions: Mobile phase: 20mM potassium phosphate (pH 2.5); Flow: 1.0mL/min.; Temp.: 30°C; Det.: UV @ 226nm

Phase collapse caused for experimental purposes by releasing column pressure

Column:

Conventional Ultra C18

Cat.#

9174565

Dimensions:

150 x 4.6 mm

Particle size:

5μm

Pore size:

100Å

LC_FF0432

LC_FF0431

LC_FF0434

LC_FF0433

1. tartaric acid

2. quinic acid

3. malic acid

4. citric acid

5. fumaric acid

Ultra Aqueous C18 Phase

Initial Analysis

No phase collapse

Phase collapse

Conventional C18 Phase