Separation of Organic Acids with 100% aqueous mobile phase using an Ascentis^® Express AQ-C18 HPLC Column

ORGANIC ACIDS IN FOODS AND THEIR ANALYSIS

Organic acids are naturally present in many food products, including fruits, vegetables, and wine. Further, they are also commonly used in the food and beverage industry as preservatives and stabilizers. Besides analytical methods involving colorimetric reaction and enzyme assays, chromatographic techniques allow simultaneous analysis of most of the organic acids. HPLC is the most popular method of choice for analyzing organic acids because of its simplicity, speed, and stability. Hence, an HPLC method was developed for the simultaneous analysis of the nine organic acids.

This application illustrates the separation of nine polar organic acids (Figure 1) on the Ascentis Express^® AQ-C18 column using a 100% aqueous mobile phase at low pH. In this method, a column length of 250 mm was selected to provide satisfactory resolution with a reasonable run time for the analysis of this mixture of polar organic acids.

CHEMICAL STRUCTURES OF ORGANIC ACIDS UNDER STUDY

Figure 1a. Chemical structure of Oxalic acid

Figure 1b. Chemical structure of Tartaric acid

Figure 1c. Chemical structure of Malic acid

Figure 1d. Chemical structure of Ascorbic acid

Figure 1e. Chemical structure of Lactic acid

Figure 1f. Chemical structure of Acetic acid

Figure 1g. Chemical structure of Citric acid

Figure 1h. Chemical structure of Succinic acid

Figure 1i. Chemical structure of Fumaric acid

EXPERIMENTAL CONDITIONS FOR THE CHROMATOGRAPHY OF ORGANIC ACIDS

HPLC Conditions
Column:	Ascentis® Express AQ-C18, 250 x 4.6 mm, 2.7 µm (577341-U)
Mobile phase:	20 mM potassium phosphate buffer, pH 2.7, isocratic
Flow rate:	1 mL/min
Back pressure:	306 bar (4452 psi)
Column temp.:	40 oC
Detector:	UV 214 nm
Injection:	20 µL
Diluent:	Mobile phase
Sample:	10 - 210 µg/mL in 20 mM potassium phosphate buffer, pH 2.7

CHROMATOGRAPHIC DATA OF ORGANIC ACIDS ANALYSIS BY HPLC-UV

Figure 2. Chromatogram showing peaks obtained for the analysis of nine Organic acids (Oxalic Acid, Tartaric Acid, Malic Acid, Ascorbic Acid, Lactic Acid, Acetic Acid, Citric Acid, Succinic Acid, and Fumaric Acid) on Ascentis® Express AQ-C18 column

Chromatographic data of organic acids in the sample

Peak No.	Compound	Concentration in the sample (µg/mL)
1	Oxalic Acid	19
2	Tartaric Acid	19
3	Malic Acid	76
4	Ascorbic Acid	19
5	Lactic Acid	210
6	Acetic Acid	210
7	Citric Acid	210
8	Succinic Acid	210
9	Fumaric Acid	10

Conclusion

The Ascentis^® Express AQ-C18 column, based on Fused-Core® particles, is a C18 bonded phase prepared using a proprietary procedure that incorporates a small amount of polar silane which makes the phase resistant to de-wetting. This allows the users of the AQ-C18 phase to run highly aqueous (up to 100%) mobile phases without the risk of phase collapse. The modified C18 phase exhibits enhanced retention of polar compounds under 100% aqueous conditions, there by efficient separation of organic acids.