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www.restekcorp.comHPLC Analysis of
Carboxylic Acids
Using Ultra Aqueous C18 Columns
Small carboxylic acids can be difficult to retain
using reversed phase high performance liquid
chromatography (HPLC) and often require a highly
aqueous mobile phase. Unfortunately, highly aque-
ous mobile phases are problematic for many C18
columns, leading to a reversible retention loss that
is attributed to chain folding. Exposure to com-
pletely aqueous mobile phases at ambient pressure
(no flow) accelerates the chain folding process.
The Restek Ultra Aqueous C18 column, however,
was designed to enhance retention of polar com-
pounds and to provide completely stable reten-
tion—even when using 100% aqueous mobile
phases. This column was compared to a conven-
tional C18 column during continual exposure to a
100% aqueous mobile phase (phosphate buffer)
Figure 3
The Ultra Aqueous C18 column demonstrates excellent reproducibility after 268 injections.
injection #1
injection #268
Sample:
tobacco extract;
Inj. vol.:
20µL;
Column:
Ultra
Aqueous C18 (cat.# 9178575)
Dimensions:
250 x 4.6mm, 5µm;
Mobile phase:
50mM potassium
phosphate, pH 2.5;
Flow:
1.0mL/min.;
Det.:
UV @ 210nm
1 2 3 4 5 6 7 min.
The Ultra Aqueous C18 column shows
remarkable stability, even after 3 days of
continuous exposure to a 100% aqueous
mobile phase.
1 2
3
4 5
6
7 min.
A conventional C18 column exhibits chain
folding after exposure for 5 minutes with
stopped flow.
1. glycolic acid, 5.4mg/mL
2. malonic acid, 4.2mg/mL
3. acetic acid, 7.8mg/mL
4. maleic acid, 0.06mg/mL
Inj. vol:
10µL;
Column:
Ultra
Aqueous C18 (cat.# 9178565);
Dimensions:
150 x 4.6mm,
5µm;
Mobile phase:
50mM
potassium phoshate, pH 2.5;
Flow:
1.0mL/min.;
Det.:
UV @ 210nm
by Terry Reid, HPLC Chemist
over a three-day period of intermittent analysis and
storage (Figure 1). While we do not recommend
storing columns with buffer, this experiment
demonstrates the complete stability of the Ultra
Aqueous C18 column against retention loss caused
by chain folding.
The analysis of four small carboxylic acids using an
Ultra Aqueous C18 column and a completely aque-
ous mobile phase shows that these polar com-
pounds can be successfully retained and resolved by
reversed phase HPLC (Figure 2). Also, the repro-
ducibility of this column is shown in the analysis of
a tobacco extract. After 268 injections, the retention
and peak shape are almost identical (Figure 3).
The unique characteristics of the Ultra Aqueous C18
column are advantageous for analyzing a wide
range of polar compounds, including carboxylic
acids. The ability to use highly aqueous mobile
phases maximizes retention of polar compounds to
provide enhanced resolution.
Figure 2
The Ultra Aqueous C18 column successfully
retains and resolves carboxylic acids.
✔
Ultra Aqueous C18 5µm HPLC Columns
For the complete HPLC columns offering, refer to Restek’s
Annual Chromatography Products Catalog
(lit. cat.# 59960).
Length
1.0mm ID 2.1mm ID
3.2mm ID
4.6mm ID
30mm
9178531
9178532
9178533
9178535
50mm
9178551
9178552
9178553
9178555
100mm
9178511
9178512
9178513
9178515
150mm
9178561
9178562
9178563
9178565
200mm
9178521
9178522
9178523
9178525
250mm
9178571
9178572
9178573
9178575
1
2
3
4
1. oxalic acid, 0.05mg/mL
2. pyruvic acid, 0.18mg/mL
3. malic acid, 0.42mg/mL
4. citric acid, 1.7mg/mL
Inj. vol:
10µL;
Column:
Ultra Aqueous C18
(cat.# 9178575);
Dimensions:
250 x 4.6mm, 5µm;
Mobile phase:
50mM potassium phoshate, pH 2.5;
Flow:
1.0mL/min.;
Det.:
UV @ 210nm
✔
Highly aqueous mobile phases
provide maximum retention
✔
Proven reproducibility under
harsh conditions
1. oxalic acid
2. pyruvic acid
3. malic acid
4. citric acid
Restek’s
New Silica
Coming soon!
✔
Highly reproducible
✔
Dependable supply
More information coming soon
to
www.restekcorp.com/hplc.htmLC_0171,
LC_0172
LC_0142
LC_0169
Figure 1
1,2,3,4