Application Note
Food Testing &
Agriculture
Author
Anne Mack
Agilent Technologies, Inc.
Abstract
This Application Note analyzed 11 organic acids with an Agilent InfinityLab Poroshell
120 Phenyl-Hexyl (2.1 × 150 mm, 2.7 µm) column using a phosphate buffer and
an acetonitrile gradient. The 11 structurally similar compounds were well resolved
within three minutes, with a minimum resolution of 1.5.
Separation of 11 Organic Acids on
an Agilent InfinityLab Poroshell 120
Phenyl-Hexyl Column with 2.7 µm
Particle Size
2
Introduction
Superficially porous particle LC
columns are a popular tool in liquid
chromatography. These columns
are more efficient at lower pressure
compared to their totally porous particle
column counterparts.¹ This is primarily
due to a shorter mass transfer distance
and substantially narrower particle
size distribution of the particles in the
column.² The most popular particle
size for superficially porous particle
columns is 2.5 to 3 µm. The larger
InfinityLab Poroshell 120 particles can
easily be used in long column formats to
maximize resolving power with UHPLC
efficiency, while not exceeding pressure
limitations.
This study demonstrates the UHPLC
performance of a long (150 mm)
InfinityLab Poroshell 120 Phenyl-Hexyl
column with a 2.7 µm particle size, and
its ability to resolve 11 closely related
compounds.
Experimental
An Agilent 1290 Infinity II LC was used
in this experiment. The system was
modified from its standard configuration
to have lower system volume and
dispersion. Table 1 shows the
configuration details. Three Agilent LC
columns were used in this experiment,
and are listed in Table 1. Table 2 shows
the LC method parameters.
Table 1. System configuration.
Agilent 1290 Infinity II LC System Configuration
Agilent 1290 Infinity II Flexible Pump (G7104A)
Degasser
Seal wash pump
35 µL solventmixer: Agilent Jet Weaver, 35 µL/100 µL (p/n G4220-60006)
Firmware: B.07.23 [0009]
Agilent 1290 Infinity II Vialsampler(G7129B)
Sample thermostat (p/n G7167-60101)
Metering parameter: seat assembly PEEK 0.12 mm, sample loop 20 µL, analytical head 20 µL
Autosampler & heater: capillary, stainless steel, 0.12 × 105 mm, SL/SL (p/n 5500-1238)
Vial, screw top, amber with write-on spot, certified, 2 mL, 100/pk (p/n 5182-0716)
Cap, screw, blue, PTFE/red silicone septa, 100/pk (p/n 5182-0717)
Vial insert, 250 µL, glass with polymer feet, 100/pk (p/n 5181-1270)
Firmware: D.07.23 [0009]
Agilent InfinityLab LC Series Integrated Column Compartment
(G7130A)
Integral type: G7129B
3.0 µL heat exchanger
Heater & column: A-Line quick-connect assembly, 105 mm, 0.075 mm (p/n 5067-5961)
Column & flow cell: capillary, stainless steel, 0.075 × 220 mm, SV/SLV (p/n 5067-4784)
Firmware: B.07.23 [0009]
Agilent 1290 Infinity II Diode Array Detector (G7117B)
Ultralow dispersion Max-Light cartridge flow cell, 10 mm, 0.60 µL (p/n G4212-60038)
UV lamp (5190-0917)
Firmware: D.07.23 [0009]
Agilent LC Columns
Agilent InfinityLab Poroshell 120 Phenyl-Hexyl, 2.1 × 150 mm, 2.7 μm (p/n 693775-912)
Agilent InfinityLab Poroshell 120 PFP, 2.1 × 150 mm, 2.7 μm (p/n 693775-408)
Agilent InfinityLab Poroshell 120 SB-Aq, 2.1 × 150 mm, 2.7 μm (p/n 683775-914)
Table 2. Method parameters.
Column
Mobile Phase
Flow Rate
(mL/min)
Mobile Phase
Composition
Injection
Volume (μL)
Sample
Thermostated Column
Compartment (°C)
Diode Array
Detector
Agilent InfinityLab
Poroshell 120
Phenyl-Hexyl,
2.1 ×150 mm, 2.7 μm
A) water
B) acetonitrile
C) n/a
D) 200 mM sodium
phosphate + 1% phosphoric
acid (85%), pH ~2
0.4
Gradient
0 to 30% B in 3 minutes,
hold 10% D throughout run,
4 minute re-equilibration
0.1
0.4 to 3 mg/mL each
in water
See Table 3 for exact
concentrations
See Figure 1 for
structures
28
210 nm, 80 Hz
3
The 11 organic acids analyzed in this
work were purchased from Sigma-Aldrich
(St. Louis, MO, USA). Figure 1 shows
their structures, and Table 3 displays
the concentrations at which they were
analyzed. Sodium phosphate and
phosphoric acid were also purchased
from Sigma-Aldrich. Acetonitrile was
purchased from Honeywell (Burdick and
Jackson, Muskegon, MI, USA). Water
was 0.2 µm filtered 18 MW from a Milli-Q
system (Millipore, Burlington, MA, USA).
Table 3. Sample composition.
Analyte
Concentration (mg/mL) in Water
Acetic Acid
2.8
t-Aconitic Acid
0.044
Citric Acid
1.3
Fumaric Acid
0.046
Lactic Acid
3.2
Maleic Acid
0.043
Malic Acid
1.4
Malonic Acid
1.3
Oxalic Acid
0.45
Succinic Acid
1.8
Tartaric Acid
0.86
Figure 1. Compounds of interest.
Acetic acid
Fumaric acid
Malic acid
Succinic acid
Tartaric acid
Malonic acid
Lactic acid
t-Aconitic acid
Citric acid
Maleic acid
Oxalic acid
O
H
3C
OH
O
O
OH
HO
O
O
OH
HO
O
O
OH
HO
O
O
O
OH
OH
OH
HO
O
O
O
OH
OH
HO
O
O
OH
HO
O
O
OH
OH
HO
O
O
OH
OH
OH
HO
O
H
3C
OH
OH
O
O
OH
HO
4
Results and discussion
Figure 2 shows three InfinityLab
Poroshell 120 columns screened for the
analysis of organic acids with a simple
aqueous isocratic method. These three
phases were chosen due to their ability
to be used with 100% aqueous mobile
phases without the risk of dewetting.
This feature can help to retain polar
compounds, such as these organic acids,
in reversed-phase LC mode. Phenyl-Hexyl
provides the best separation of the early
eluting acids, with the Agilent InfinityLab
Poroshell 120 SB-Aq as a close second.
Unfortunately, the SB-Aq phase struggles
to separate malic acid and lactic acid.
Figure 2. Alternate stationary phases on Agilent InfinityLab Poroshell 120 columns for organic acids.
A: water, D: 200 mM sodium phosphate + 1% phosphoric acid (85%), pH ~2; isocratic: 90% A/10% D;
0.4 mL/min; 20 °C; 210 nm; 2.1 × 150 mm, 2.7 µm Agilent InfinityLab Poroshell 120 columns.
x10²
-0.1
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
Acquisition time (min)
1.2
1.4
1.6
1.8
2.0
2.2
2.4
2.6
2.8
3.0
3.2
3.4
3.6
3.8
4.0
PFP
Tartaric
Malic
Lactic
Acetic
Citric
Fumaric
Response units (%
)
x10²
-0.1
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2.0 2.1 2.2 2.3 2.4 2.5 2.6
Acquisition time (min)
SB-Aq
Tartaric
Malic
Lactic
Acetic
Maleic
Citric
Fumaric
t-Aconitic
Response units (%
)
x10²
-0.1
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
Acquisition time (min)
0.9
1.0
1.1
1.2
1.3
1.4
1.5
1.6
1.7
1.8
1.9
Phenyl-Hexyl: best separation of early eluting acids
Tartaric
Malic
Lactic
Acetic
Maleic
Citric
Fumaric
Response units (%
)
5
The partial coelution of citric and
fumaric acid on Phenyl-Hexyl is solved
by increasing the temperature of the
column, as demonstrated in Figure 3.
Figure 4 shows the final separation of
11 organic acids on a 2.1 × 150 mm,
2.7 µm InfinityLab Poroshell 120
Phenyl-Hexyl column. All compounds are
well resolved, with a minimum resolution
of 1.5, in three minutes. The organic
acids are difficult to retain because
they are very polar, and are also difficult
separate because they are structurally
similar (Figure 1). However, the long,
150 mm, 2.7 µm Phenyl-Hexyl column
has sufficient efficiency and resolving
power to successfully retain and
separate this sample.
Figure 3. Column temperature screen for organic acids on Agilent InfinityLab Poroshell 120 Phenyl-Hexyl
column.
x101
0
1
2
x10¹
0
1
2
x10¹
0
1
2
Acquisition time (min)
0.1
0.3
0.5
0.7
0.9
1.1
1.3
1.5
1.7
1.9
2.1
2.3
2.5
2.7
2.9
Response units (%
)R
esponse units (%
)
Response units (%
)
20 °C
30 °C
40 °C
×102
-0.1
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
Acquisition time (min)
0.1
0.3
0.5
0.7
0.9
1.1
1.3
1.5
1.7
1.9
2.1
2.3
2.5
2.7
2.9
Response units (%
)
1. Oxalic acid
2. Tartaric acid
3. Malic acid
4. Malonic acid
5. Lactic acid
6. Acetic acid
7. Maleic acid
8. Citric acid
9. Fumaric acid
10. Succinic acid
11.
t-Aconitic acid
LC method is as shown in Table 2
1
2
3 4
5
6
7
8
9
10
11
Figure 4. Separation of 11 organic acids on an Agilent InfinityLab Poroshell 120 Phenyl-Hexyl column.
www.agilent.com/chem
This information is subject to change without notice.
© Agilent Technologies, Inc. 2019
Printed in the USA, June 26, 2019
5994-1082EN
Conclusion
The Agilent InfinityLab Poroshell 120,
2.7 μm column is used to accomplish
a challenging separation of 11 organic
acids. The high efficiency of this
superficially porous particle column
provides enough resolution to resolve
the closely related compounds on a
2.1 × 150 mm column.
References
1. Gratzfield-Hugsen, A.; Naegele, E.
Maximizing Efficiency Using
Agilent InfinityLab Poroshell 120
Columns. Agilent Technologies
Application Note, publication number
5990-5602EN, 2016.
2. Meyer, V. R. Practical
High-Performance Liquid
Chromatography. Fourth Edition,
p. 34. Wiley, 2004.
