HILIC and Mixed‐Mode Retention of an Embedded‐Polar Stationary Phase
Sharon Lupo, Ty Kahler, Jason Thomas, Chris Denicola, Rick Lake; Restek Corporation
ABSTRACT
Figure 3: Effect of Temperature on Analysis of Benzalkonium Chloride and Neutral Compounds by Reversed‐Phase
HILIC and Mixed‐Mode are terms originally used to describe a specific chemical interaction or set of interactions in liquid chromatography. Recently, however, these terms have been more frequently used to describe LC columns offering retention mechanisms that vary, or are orthogonal, to a C18. These columns generally consist of a bonded phase which contains a chemical moiety other than, or in addition to, alkyl carbon chains. By incorporating double bonds, oxygen, nitrogen, or other heteroatoms, alternate retention and selectivity can be accomplished. These chemical interactions are generally not well understood or easily demonstrated, which can leave users frustrated and not using the phase to its potential.
5
°C
1
3
Overall
Retention Loss
50
1: C12
2: Benzene
3: C14
4: Toluene
5: Naphthalene
6: Biphenyl
4
2
6
BACKGROUND
When comparing the results of the Hydrophobic‐Subtraction Model [1] between an IBD and a standard C18 phase on the same silica , it can be determined that in addition to hydrophobic retention, the IBD phase is also capable of being a very strong hydrogen‐
bond acceptor, (B‐term in Figure 1). This interaction may be so strong as to be labeled as anion‐exchange. For a full list of terms and definitions please refer to Reference 1
2
1
H
S*
A
B
Ultra C18
Ultra IBD
C
C‐7
1.051
0.033
‐0.032
0.672
‐0.035
‐0.052
‐0.023
0.057
‐0.003
0.233
‐0.564
0.860
Figure 2: Example Structures of Benzalkonium Chloride and Neutral Compounds Used
in IBD Study
+
2
10
Time (min)
0
30
4
Time (min)
60
0
In the next example, ethyl sulfate (ETS) was chosen to demonstrate the retention behavior of the IBD stationary phase under mixed‐mode or HILIC conditions (Figure 6). ETS is a strong acid which exhibits a negative charge in solution and is capable of anion
ETS is a strong acid which exhibits a negative charge in solution and is capable of anion‐
exchange interactions. 1 – Benzene
2 – C12
3 – Toluene
3 4 – C14
5 – Naphthalene
6 ‐ Biphenyl
3
4
1
6
1.0
Figure 9: Effect of Temperature on Analysis of ETS by Anion‐Exchange
Figure 9: Effect of Temperature on Analysis of ETS by Anion
Exchange Mechanism
Mechanism
°C
O
H3C
5
O
S
O
-
30
Ethyl Sulfate
(ETS)
Increased Temperature
Increased Retention
O
When analyzed, ETS shows the greatest reversed‐phase retention with the use of a 100% aqueous mobile phase. However, retention is also achieved with increased organic based on the anion‐exchange mechanism of the IBD phase. (Figure 7)
60
0
5
3
1
4
6
U‐Shaped Retention Indicative
Of Multi‐Modal Retention Mechanism %B
5
0
1
0.1
0
4
3
Time (min)
Ultra IBD 150 x 4.6 mm, 5µm; 1 mL/min; 30°C; 207 nm
80% A: formic acid / Water; 20% B: formic acid / Methanol
55%B to 95% B over 10 min
6
10
k
35
Ultra IBD 50 x 2.1 mm, 3µm; 0.4 mL/min
40% A: 0.1% formic acid / Water
60% B: 0.1% formic acid / Acetonitrile
Figure 7: Analysis of ETS by Reversed Phase and by Anion‐Exchange Mechanism
2
0.5
15
Ultra IBD 50 x 2.1 mm, 3µm; 0.4 mL/min
80% A: 0.1% formic acid + 2mM NH4‐formate / Water
20% B: 0.1% formic acid + 2mM NH4‐formate /Acetonitrile
Figure 6: Structure of ETS Used in IBD Study
Figure 4: Effect of Acid Concentration on Analysis of Benzalkonium Chloride and Neutral Compounds by Reversed‐Phase
Increased Retention of Ionic Compounds
Ionic Compounds
k
METHOD DEVELOPMENT – Ethyl Sulfate (ETS)
2
The initial analysis revealed a coelution between one of the benzalkonium analogs and the neutral benzene. An increase in temperature is typically accompanied by a decrease in retention in reversed‐phase chromatography. However, it may also result in a change of selectivity for ionic compounds such as benzalkonium chlorides (Figure 3) As the temperature was increased greater selectivity was observed for the two
3). As the temperature was increased greater selectivity was observed for the two closely eluting compounds. Unfortunately, the resulting resolution was insufficient.
Increased Temperature
Decreased Retention
10
Ultra IBD 150 x 4.6 mm, 5µm; 1 mL/min; 30°C; 207 nm 80% A: formic acid / Water; 20% B: formic acid / Methanol
55%B to 95% B over 10 min
The ionic interactions exhibited by the embedded polar group of the IBD stationary phase may be influence by both the acid concentration as well as the strength of the specific acid utilized. This mechanism is demonstrated in the following example, increased acid concentration results in increased retention for the benzalkonium chloride compounds while the neutrals are unaffected (Figure 4). When a stronger acid, such as TFA, is utilized, the retention for the ionic compounds increases dramatically (Figure 5) . Since the retention of the neutral compounds remains constant regardless of concentration or strength, adjusting the acid enables the user to optimize the selectivity of the IBD phase.
H3C
Toluene
Figure 8: Effect of Temperature on Analysis of ETS by Reversed‐ Phase
°C
6
1
Ultra IBD 150 x 4.6 mm, 5µm; 1 mL/min; 30°C; 207 nm
A: 0.1% H3PO4 /Water; B: Acetonitrile
35%B to 85% B over 10 min
CH3
Benzalkonium Chloride (C12 analog)
6
3
1.0%
TFA
6
CH3
CH3
N
– Benzene
– C12
– Toluene
– C14
– Naphthalene
- Biphenyl
Similar to the benzalkonium chloride example, an increase in temperature in reverse‐
phase chromatography causes a decrease in the retention of ETS. (Figure 8). However, when utilizing anion‐exchange retention mechanism temperature can often have a dramatically different effect. In this case, the effect of temperature on the anion‐
exchange mechanism serves to increase retention (Figure 9).
5
2
In this example a mixture of benzalkonium chlorides (C12 and C14) and several neutral compounds (Figure 2) were chosen to demonstrate the retention behavior of the IBD stationary phase under reversed‐phase conditions. The benzalkonium chlorides consist of even‐numbered alkyl chain lengths that are capable of hydrophobic retention. In addition, the quaternary ammonium group maintains a formal positive charge capable of ionic interactions.
4
4
30
% A id
Acid
METHOD DEVELOPMENT ‐ BENZALKONIUM CHLORIDES
3
3
0
1
2
3
4
5
6
2
1
1.0%
FA
Figure 1: Hydrophobic‐Subtraction Model Values for IBD Compared to C18 on Identical Silica Column
5
2‐Fold Increase
In Retention
5
Increased Resolution
In this study we set out to explore the retention mechanisms of the Restek IBD stationary phase. This proprietary phase offers unique selectivity by embedding a polar group into an alkyl chain which takes a positive charge under selected conditions. In addition to the traditional reversed‐phase dispersive interactions, this diti
I dditi t th t diti
l
d h
di
i i t
ti
thi
phase may exhibit hydrogen bonding and anion‐exchange interactions which aid in selectivity of specific analytes. Our focus in this presentation is on specific method parameters including temperature, and mobile phase acid strength and concentration and how these changes affect the selectivity of target analytes. By demonstrating the influence of specific method changes on this phase we aim to gather a better understanding of the interactions demonstrated by the IBD phase and its use as a HILIC or mixed‐mode phase.
Figure 5: Effect of Acid Strength on Analysis of Benzalkonium Chloride and Neutral Compounds by Reversed‐Phase
CONCLUSIONS
The IBD allows retention based on hydrophobicity, ionic interactions, and anion‐
exchange exchange
20
Temperature may be used to significantly alter selectivity of the IBD phase. Temperature may affect the selectivity of the phase in opposite ways depending on the driving separation mode
40
Acid strength and concentration can be used to alter selectivity based on the ionic interactions between the IBD phase and the solutes
60
0
k
Ultra IBD 50 x 2.1 mm, 3µm; 0.4 mL/min
Ult
IBD 50 2 1
3
0 4 L/ i
A: 0.1% formic acid/Water
B: 0.1% formic acid/Acetonitrile
30
The flexibility of the IBD phase makes it a good choice for customizable selectivity and retention of quaternary amines and strong acids [[1] L.R. Snyder, J.W. Dolan, P.W. Carr, The Hydrophobic‐Subtraction Model of Reversed‐Phase ]
y ,
,
,
y p
Column Selectivity, J. Chromatogr. A 1060 (2004) 77.