APPLICATION BRIEF

Liquid Chromatography

Author:
Wilhad M. Reuter
PerkinElmer, Inc.
Shelton, CT

The Analysis of a Broad

Range of Organic Acids by Introduction
Organic acids are prevalently
HPLC with UV Detection found in all fruit juices and
many other beverages. As
they are known to impart
particular taste and/or aromatic qualities to such drinks, both pleasant and
unpleasant, there is considerable interest in monitoring organic acids during
production. With this in mind, this application brief provides a routine and
reproducible method for the HPLC analysis of 11 organic acids commonly
found in juices and other beverages, focusing especially on the relatively
higher molecular weight acids.
 Experimental Table 1. LC Method Parameters.
PerkinElmer Brownlee™ Aqueous C18 5-µm, 250 x 4.6-mm
Hardware/Software Column:
(Part# N9303549)
For the chromatographic separations, a PerkinElmer Altus™ HPLC Mobile Phase A: 10-mM KH2PO4, pH 2.4 with phosphoric acid
System was used, including the Altus A-10 Solvent/Sample Mobile Phase B: Acetonitrile (ACN)
Solvent program:
Module, integrated vacuum degasser, A-10 column heater, and
Time Flow Rate
Altus A-10 UV detector. All instrument control, analysis and data Mobile Phase: (min) (mL/min)
%A %B Curve

processing was performed using the Waters® Empower® 3 1 Initial 1.5 80.0 20.0
2 10.0 1.5 40.0 60.0 6
Chromatography Data Software (CDS) platform. 3 12.5 1.5 40.0 60.0 6
4 12.6 1.5 80.0 20.0 11
Method Parameters
The LC method parameters are shown in Table 1. Analysis Time: 12.5 min.; 5-min. injection delay time (re-equil.)
Flow Rate: 1.5 mL/min.
Solvents, Standards and Samples
Pressure: 2650 psi/177 bar (maximum)
All solvents, reagents, and diluents used were HPLC-grade and
Oven Temp.: 30 ºC
filtered via 0.45-µm filters. For all dilutions, HPLC-grade water Detection: 210 nm
was used. Injection Volume: 20 µL
The following standards were obtained from Sigma-Aldrich®, Inc Sampling (Data) Rate: 5 pts./sec
(Allentown, PA): lactic acid, acetic acid, butyric acid, isobutyric acid,
propionic acid, valeric acid, isovaleric acid, methylvaleric acid, Results and Discussion
heptanoic acid, hexanoic acid and octanoic acid.
Using the optimized chromatographic conditions described
A 1000-ppm working standard solution was prepared by adding above, Figure 2 shows the HPLC separation of the level-4
50 µL of each organic acid standard to a 50-mL volumetric cylinder (250-ppm) working standard containing the eleven organic
and filling to volume with diluent. Six calibration levels were acids. All analytes were well resolved, except for butyric and
prepared via serial dilution of the working standard solution. Each isobutyric acids, which coeluted at 4.5 minutes.
standard level was injected in duplicate.

0.024

0.022

0.020
Butyric/Isobutyric

0.018
Lactic

0.016
Acetic

Propionic

0.014

0.012
Methylvaleric
Isovaleric
AU

Valeric

0.010
Hexanoic

Heptanoic

0.008

0.006
Octanoic

0.004

0.002

0.000

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00
Minutes

Figure 1. Chromatogram of the level-4 (250-ppm) working standard, containing 11 organic acids; by UV at 210 nm.

2
Figure 2 shows the overlay of 12 replicate injections of the level-4 (250-ppm) working standard, demonstrating high reproducibility.
The retention time precision for all analytes was < 0.075% RSD.

0.020

0.015

0.010
AU

0.005

500000

0.000
400000

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00
Minutes
300000
Area

Figure 2. Overlay of 12 replicates of level-4 (250-ppm) working standard, by UV at 210 nm. 200000

100000

Figure 3 shows the calibration results for two representative analytes in the range of 25–1000 ppm. As butyric and isobutyric acids
0
coeluted, the combined concentration range was 50-2000 ppm. All organic acids showed an exceptional linear fit, with R2 values >
0.9999 (n = 2 at each level) for all analytes. -100000
0.00 100.00 200.00 300.00 400.00 500.00 600.00 700.00 800.00 900.00 1000.00
Concentration (ppm)

500000
Lactic acid 250000
Acetic acid
400000
200000

300000
150000
Area

Area

200000 100000

100000 50000

0 0

R2 = 0.99992 R2 = 0.99994
-100000 -50000
0.00 100.00 200.00 300.00 400.00 500.00 600.00 700.00 800.00 900.00 1000.00 0.00 100.00 200.00 300.00 400.00 500.00 600.00 700.00 800.00 900.00 1000.00

Concentration (ppm) Concentration (ppm)

Figure 3. Results of 6-level calibration set for lactic and octanoic acids.
250000

200000

150000
Area

100000

50000

-50000
0.00 100.00 200.00 300.00 400.00 500.00 600.00 700.00 800.00 900.00 1000.00

Concentration (ppm)

3
 Table 2 presents the calculated limits of detection and quantitation Conclusion
(LOD, LOQ) for the analyzed organic acids. These limits were The results obtained confirm the applicability of this method for
derived using the signal-to-noise (s/n) results obtained during the effective and robust chromatographic analysis of a broad
calibration. The LODs and LOQs ranged from 0.5-1.8 and 1.4- range of organic acids. Apart from the co-elution of butyric
6.0 ppm, respectively. and isobutyric acids, the analytes were well separated in under
Table 2. Calculated LOQ and LOD values for the eleven organic acids. 12 minutes by HPLC using UV detection. The results showed
excellent retention time repeatability as well as exceptional
LOD (ppm) LOQ (ppm)
Organic Acids linearity over the tested concentration range.
(s/n ≥ 3/1) (s/n ≥ 10/1)
lactic 0.5 1.4
acetic 0.5 1.4
butyric/isobutyric 0.6 2.1
propionic 0.6 2.1
valeric 0.9 2.8
isovaleric 0.7 2.6
methylvaleric 1.0 3.5
heptanoic 1.1 3.7
hexanoic 1.0 3.5
octanoic 1.8 6.0

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