share:
article_image

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 AllureAqueous 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. The Ultra Aqueous C18 phase resolves organic acids in a 100% aqueous mobile phase without loss of retention. In comparison, the conventional C18 phase 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.

PeaksConc.
(µg/mL)
1.Tartaric acid2,000
2.Quinic acid2,000
3.Malic acid2,000
4.Citric acid2,000
5.Fumaric acid10
Initial Analysis on Ultra Aqueous C18
LC_FF0433
PeaksConc.
(µg/mL)
1.Tartaric acid2,000
2.Quinic acid2,000
3.Malic acid2,000
4.Citric acid2,000
5.Fumaric acid10
No phase collapse on Ultra Aqueous C18
LC_FF0434
PeaksConc.
(µg/mL)
1.Tartaric acid2,000
2.Quinic acid2,000
3.Malic acid2,000
4.Citric acid2,000
5.Fumaric acid10
No phase collapse on Ultra Aqueous C18
LC_FF00431
PeaksConc.
(µg/mL)
1.Tartaric acid2,000
2.Quinic acid2,000
3.Malic acid2,000
4.Citric acid2,000
5.Fumaric acid10
No phase collapse on Ultra Aqueous C18
LC_FF0432
ColumnConventional Ultra C18 (cat.# 9174565)
Dimensions:150 mm x 4.6 mm ID
Particle Size:5 µm
Pore Size:100 Å
Temp.:30 °C
SampleFruit Juice Organic Acid Standard (cat.# 35080)
Diluent:deionized water
Inj. Vol.:10 µL
Mobile Phase20 mM potassium phosphate (pH 2.5)
Flow:1.0 mL/min
DetectorUV/Vis @ 226 nm
InstrumentShimadzu Prominence


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

PeaksConc.
(mg/mL)
1.Tartaric acid1.0
2.Quinic acid1.0
3.Malic acid1.0
4.Citric acid1.0
5.Fumaric acid0.005
Organic Acids Standard on Allure Organic Acids
LC_0238
ColumnAllure Organic Acids (cat.# 9165585)
Dimensions:300 mm x 4.6 mm ID
Particle Size:5 µm
Pore Size:60 Å
Temp.:ambient
Samplestandard solution
Diluent:water
Inj. Vol.:10.0 µL
Mobile Phase100 mM phosphate buffer, pH 2.5
Flow:0.5 mL/min
DetectorUV/Vis @ 226 nm

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

Peaks
1.Quinic acid
2.Malic Acid
3.Citric Acid
Organic Acids in Cranberry Juice on Allure Organic Acids
LC_0236
ColumnAllure Organic Acids (cat.# 9165585)
Dimensions:300 mm x 4.6 mm ID
Particle Size:5 µm
Pore Size:60 Å
Temp.:ambient
Samplecranberry juice:water (50:50, v/v)
Diluent:water
Inj. Vol.:10.0 µL
Mobile Phase100mM phosphate buffer, pH 2.5
Flow:0.5 mL/min
DetectorUV/Vis @ 226 nm

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

RELATED SEARCHES

AOAC method 986.13

,

Allure Organic Acids

,

cranberry juice

,

fruit juice

,

Ultra Aqueous C18

Restek Domestic Customer Service

Subject

Message

Your Full Name

Your Email

Company Name

Address

Spam Block (Please leave this blank)

all fields required

Thank you

Your message has been sent. We will be in touch shortly.

Message not sent

Sorry, your message could not be sent at this time. Please try again later, or contact Restek or your local Restek representative via phone.

www.restek.com/Contact-Us