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Food, Flavors, & Fragrances Article

Simple, Reliable HPLC Analyses of Organic Acids

Using Water-Compatible Allure® or Ultra C18 Columns

by 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 organic acids, including quinic and tartaric.

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 because it is easy to use. 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 the mobile phase of choice. A low pH buffer is used to ensure that all acidic groups are protonated (otherwise, the organic acids are 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—were designed using aqueous-compatible C18 phases that combat phase collapse. 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 comparison, 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.

In analyses of organic acids, specifically, under high aqueous mobile phase conditions, the Allure® Organic Acids column is the column of choice. We have developed a method using a 300mm Allure® Organic Acids column to separate critical organic acids: tartaric, quinic, malic, citric and fumaric acids. This method uses 100% aqueous mobile phase as recommended by AOAC method 986.13. In addition to allowing repeated injections in 100% aqueous mobile phase without the severe lose of retention observed with conventional C18 column technology, the Allure® Organic Acids column is prepared and tested specifically for separating organic acids. Figure 2 shows that tartaric and quinic acids are resolved to baseline; Figure 3 shows typical analyses under the conditions we recommend.


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.

  1. tartaric acid
  2. quinic acid
  3. malic acid
  4. citric acid
  5. fumaric acid
Ultra Aqueous C18 Phase
LC_FF0433, LC_FF0434

Column:
Ultra Aqueous C18 Cat.# 9178565
Dimensions:
150 x 4.6 mm
Particle size:
5µm
Pore size:
100Å

Conventional C18 Phase
LC_FF0431, LC_FF0432

Column:
Conventional Ultra C18 Cat.# 9174565
Dimensions:
150 x 4.6 mm
Particle size:
5µm
Pore size:
100Å
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:
Instrument:
Shimadzu Prominence HPLC
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


Figure 2  Excellent resolution of organic acids, including tartaric and quinic acids, on an Allure® Organic Acids column.

Peak List:
Conc.
(mg/mL)
  1. tartaric acid
  2. quinic acid
  3. malic acid
  4. citric acid
  5. fumaric acid
1
1
1
1
      0.005

Sample:
Inj.:
10µL standard solution
Solvent:
water
Column:
Allure® Organic Acids
Cat. #:
9165585
Dimensions:
300 x 4.6mm
Particle size:
5µm
Pore size:
60Å
Conditions:
Mobile phase:
100mM phosphate buffer, pH 2.5
Flow:
0.5mL/min.
Temp.:
ambient
Det.:
UV @ 226nm

LC_0238

Figure 3  Sharp, easily differentiated organic acid profiles for cranberry juice cocktail on an Allure® Organic Acids column.

  1. quinic acid
  2. malic acid
  3. citric acid

Sample:
Inj.:
10µL
Conc.:
cranberry juice:water (50:50, v/v)
Solvent:
water
Column:
Allure® Organic Acids
Cat. #:
9165585
Dimensions:
300 x 4.6mm
Particle size:
5µm
Pore size:
60Å
Conditions:
Mobile phase:
100mM phosphate buffer, pH 2.5
Flow:
0.5mL/min.
Temp.:
ambient
Det.:
UV @ 226nm

LC_0236

References

  1. Verify Fruit Juice Quality from Organic Acid Profiles from The Restek Advantage, 2003, vol. 3
  2. Official Methods of Analysis (2000). AOAC International, 17th edition, method # 986.13
  3. Manolaki, P. et al., Food Chemistry, 98 (2006), page 658-663
  4. Kafkas, E. et al., Food Chemistry, 97 (2006), page 732-736