Appl icat ion News

LAAN-A-LC-E166

No.L386

High Speed, High Resolution Analysis (Part 26)Analysis of Phenolic Acids

High Per formance Liquid Chromatography

Phenolic acid exists in higher plants as esters, ethers or in its free state, and is a compound that has been receiving increased attention in recent years due to its antioxidant effects. HPLC is often used for quantitative analysis of phenolic acid in processed foods containing fruits and fruit materials, but the analysis is often quite time-consuming because a relatively long column and

gradient elution are required to achieve separation of the contaminants in actual samples.Here we present an example of high speed, high resolution analysis of phenolic acids using the Shimadzu Prominence UFLCXR ultra high-speed LC system with the SPD-M20A photodiode array detector.

■ Simultaneous Analysis of 11 Phenolic Acids and Benzoic AcidFig. 1 shows a chromatogram of a standard mixture of 11 phenolic acids and benzoic acid (50 mg/L each), analyzed using a Shim-pack XR-ODS II high speed, high resolution column, and Table 1 shows the analytical conditions. For comparison, data acquired using a conventional Shim-pack VP-ODS column are also shown.

Table 1 Analytical Conditions

ColumnMobile Phase

Flow RateColumn Temp.Injection VolumeDetectionUV Cell

: Shim-pack XR-ODS II (100 mm L. × 3.0 mm I.D., 2.2 µm): A: 50 mmol/L ammonium formate buffer (pH3.6) B: Methanol B Conc. 20% (0-10 min)→80% (10.01-11 min)→

20% (11.01-15 min) 0.5 mL mixer: 0.9 mL/min: 40 ˚C: 4 µL: SPD-M20A (Max plot 230-350 nm): Semi-micro cell

Fig. 1 Chromatograms of a Standard Mixture of 11 Phenolic Acids and Benzoic Acid (50 mg/L each)

200

mAU

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mAU

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0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 min

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Shim-pack VP-ODS (250 mm L. × 4.6 mm I.D., 4.6 µm)Injection Volume: 10 µL, UV Cell: Conventional cell

■ Peaks1. Gallic acid, 2. Protocatechuic acid, 3. Chlorogenic acid, 4. p-Hydroxybenzoic acid, 5. Vanillic acid, 6. Caffeic acid, 7. Syringic acid8. Salicylic acid, 9. p-Coumaric acid, 10. Ferulic acid, 11. Benzoic acid, 12. Sinapic acid

Shim-pack XR-ODS II (100 mm L. ✕ 3.0 mm I.D., 2.2 µm)

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3

4

5

6

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No.L386

SHIMADZU CORPORATION. International Marketing Division3. Kanda-Nishikicho 1-chome, Chiyoda-ku, Tokyo 101-8448, Japan Phone: 81(3)3219-5641 Fax. 81(3)3219-5710Cable Add.:SHIMADZU TOKYO

Printed in Japan 3295-12902-10A-IK

NOTES:✽ This Application News has been produced and edited using information that was available when the

data was acquired for each article. This Application News is subject to revision without prior notice.

Coffee beans are known to contain a large amount of chlorogenic acid, and analysis of chlorogenic acid in coffee was introduced in Application News L306. Here we present a high speed analysis of chlorogenic acid in commercially available fruit juices.

■ Analysis of Chlorogenic Acid

Ellagic acid is found in many plants, in which much of it exists in the form of tannin combined with gallic acid. Here we analyzed free ellagic acid in processed food.

■ Analysis of Ellagic Acid

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Fig. 3 Chromatograms of Berry Juice (upper) and Peach Juice (lower)

■ Peak1. Chlorogenic acid

Berry Juice

Filtration (0.2 µm)

HPLC

Peach Juice 1 mL

Filtration (0.2 µm)

HPLC

Water 4 mL

OH

OH

O

OH

HO

OHHOOC

O

Fig. 2 Structure of Chlorogenic Acid

Table 2 Analytical Conditions

ColumnMobile Phase

Flow RateColumn Temp.Injection VolumeDetectionUV Cell

: Shim-pack XR-ODS (50 mm L. × 3.0 mm I.D., 2.2 µm): A: 50 mmol/L ammonium acetate buffer (pH4.7) B: Methanol B. Conc. 15% (0-0.8 min)→80% (0.81-1.0 min)→

15% (1.01-1.8 min): 0.9 mL/min: 40 ˚C: 4 µL: SPD-M20A (325 nm): Semi-micro cell

Fig. 4 Structure of Ellagic Acid

Table 3 Analytical Conditions

ColumnMobile Phase

Flow RateColumn Temp.Injection VolumeDetectionUV Cell

: Shim-pack XR-Phenyl (50 mm L. × 3.0 mm I.D., 2.2 µm): A: 10 mmol/L oxalic acid aq. B: Methanol/Acetonitorile = 7/3 (v/v) B. Conc. 35%: 1.0 mL/min: 40 ˚C: 2 µL : SPD-M20A (366 nm): Semi-micro cell

O

O

OH

OO

HO

HO

OH

Fig. 5 Chromatograms of Raspberry Jam (upper) and Red Wine (lower)

■ Peak1. Ellagic acid

Red Wine

Filtration (0.2 µm)

HPLC

Jam 1 g

Homogenization

Ultrasonic Extraction(30 min)

Methanol 4 mL

Filtration (0.2 µm)

HPLC

7.5

mAU

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