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surface.Active silanols can enhance retentionof polar com-
pounds, but also can cause severepeak tailing for basic com-
pounds likepyridine.The comparison inFigure4 shows that the
UltraAqueousC18 column achieves enhanced retention for polar
compoundswithout sacrificingbasedeactivation.This combina-
tionof benefits sets theUltraAqueousC18 column apart from
conventionalC18columns.
MaximumRetentionWithoutDerivatizationor Ion
PairingReagents
Aminoacids
frequently arederivatized inpreparation for
reversedphaseHPLCanalysis.Derivatizationprovides adequate
retentionand improveddetection sensitivity for thesecom-
pounds. TheUltraAqueousC18 column retains and separates
many amino acidswithout derivatizationor ionpairing reagents
(Figure 5).The amino acids in this example are three of themost
hydrophobicbecauseof their aromatic sidegroups, and thus they
arenot as difficult to retain as someof themorehydrophilic
amino acids.However, pleasenote the initialmobilephase
contains 5% acetonitrile. For themorepolar, hydrophilic amino
acids, retention canbe increased considerablyby reducing the
proportionof acetonitrile.Tomaximize retention, the acetonitrile
canbeeliminatedwithout compromising reproducibility.
Thegroupof compounds known as
water-solublevitamins
is
chemicallyverydiverse, includingboth acidic andbasic
molecules. Someof thewater-solublevitamins, such as thiamin
and ascorbic acid, areverypolar and thus difficult to retainby
reversedphaseHPLC.Many reversedphasemethods forwater-
solublevitamins require ionpairing reagents inorder to retain
themorepolar analytes. TheUltraAqueousC18 column
analyzes sixwater-solublevitaminswithgradient elution and
relatively simplemobilephases that containno ionpairing
reagents (Figure6).
PeakList
Conc.
(mg/mL)
1. tyrosine
0.2
2. phenylalanine
1.6
3. tryptophan
0.4
Column:
UltraAqueousC18Column
Catalog#:
9178565
Dimensions:
150x4.6mm
Particle size:
5µm
Pore size:
100Å
Sample:
Inj.:
20µL
Solvent:
mobile phaseA
Conditions:
Mobile phases: A: 50mM potassium phosphate, pH 2.5
B: acetonitrile
5% - 20%B: 0-5min.
20% - 5%B: 5-6min.
Hold at 5%B: 6-13min.
Flow:
1.0mL/min.
Temp.:
30°C
Det.:
UV@254nm
Figure5
TheUltraAqueousC18 column retains and separates
many amino acidswithout derivitization or
ionpairing reagents.
Conclusion
TheUltraAqueousC18 columnprovides selectivity similar to a
conventionalC18 column,whilemaintaining ahigh level of base
deactivation. In addition, theUltraAqueousC18 column can
benefit your analysis by allowing100% aqueousmobilephases;
providing enhanced retentionof polar compounds, better peak
shape, andnoneedof samplederivatizationor ionpairing
reagents.
Figure6
TheUltraAqueousC18 column separates sixwater-
soluble vitamins usinga relatively simplemobile phase
and no ion pairing reagents.
LC_0141
PeakList:
Conc.
(µg/mL)
1. thiamin (B1)
250
2. ascorbic acid (C)
1,000
3. unknown
n/a
4. nicotinic acid (B3)
1,000
5. unknown
n/a
6. pantothenic acid (B5) 1,000,000
7. folic acid (B9)
500
8. riboflavin (B2)
250
9. methyl paraben
0.2
Initial dilutions ofB1 andB2
basifiedwith ammoniumhydroxide
1
2
3
LC_0146
0
2
4
6
8
10 min.
Column:
UltraAqueous C18
Catalog #:
9178575
Dimensions: 250 x 4.6mm
Particle size: 5µm
Pore size:
100Å
Sample:
Inj.:
1µL
Solvent:
all analytes dissolved
inHPLC-gradewater
Conditions:
Mobile phase: A: 25mM potassium
phosphate, pH 2.00:
methanol (95:5)
B: methanol:25mM
potassium phosphate,
pH 3.5 (60:40)
Hold0%B: 0-6min
Step to25%B: 6.01min
25-100%B: 6.01-11min
Hold100%B: 11-16min
Flow:
1.0mL/min.
Temp.:
27°C
Det.:
UV@ 254nm
1
2
3
4
5
8
7
6
9
0 2 4 6 8 10 12 14min.
