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Authors: T.J. Higley, Tosoh Bioscience LLC, Tatsunari Yoshida, Tosoh Corporation
Abstract:
The TSKgel Amide-80 column provides excellent selectivity for separating
peptides in hydrophilic interaction chromatography due to polar carbamoyl
functionalities bonded to a silica base material.
Introduction:
The separation of peptides with many acidic and basic residues has always
proved to be problematic with RPLC gradient elution runs common in LC/MS
and/or high throughput methodologies. Several options are available to
increase retention of polar peptides including mobile phase adjustment(1)
or switching to a polar adsorptive mechanism used in both normal phase
and hydrophilic interaction chromatography (HILIC)(2,3). HILIC methods
are often advantageous because aqueous and polar organic mobile phase
systems, common to RPLC, are acceptable. The data presented within will
highlight the rational, recovery and reproducibility associated with a
HILIC method developed within Tosoh laboratories for the separation of
peptides.
Experimental Conditions:
A 5m TSKgel Amide-80 column (4.6mm ID x 25cm) was used in conjunction
with a Tosoh HPLC system. The peptides were purchased from the Peptide
Institute (Osaka, Japan) and Sigma (St. Louis, MO). Final mobile phase
conditions: Eluent A (initial eluent) contained 0.1% trifluoroacetic acid
(TFA) in acetonitrile (ACN): water (97:3) and Eluent B contained 0.1%
TFA in ACN-water (55:45). The peptides were dissolved in 10L of
ACN-water-formic acid (5:45:50) and subsequently diluted to a final volume
of 50L with Eluent A. A linear gradient
from A to B was administered
at 1.0mL/min over 70 minutes at 0.6% water/min. UV Detection was monitored
at 265nm.
Results:
In the study to find suitable method conditions for the separation of
peptides by HILIC, the effect of acid and ACN concentration were examined.
It is commonly understood that residual silanols, present with functionalized
silica based materials, can interact with the ionic residues of peptides.
This interaction affects recovery and/or causes peak tailing. For this
reason, TFA, acetic acid, formic acid and no acid were investigated to
determine the appropriate acid strength for eliminating IEX interactions.
As shown in Figure 1, elution order and peak shape were affected
differently depending the acid investigated. Without the addition of acid,
three of the peptides failed to elute and tailing was evident on the remaining
peptides. The addition of weak acids (acetic or formic) allowed the elution
of all components but did not eliminate tailing. TFA at 0.1% was strong
enough to eliminate tailing and sequester the apparent IEX interactions.
Subsequent work (not shown) indicates little improvement with the addition
of equal molar amounts of TFA and TEA at 0.1% or 0.2% over what is possible
with TFA alone(4). The advantage of using TFA to eliminate IEX interactions
is the need for desalting is eliminated when a volatile mobile phase is
used. PolyHydroxyethyl A, {poly (2-hydroxyethyl aspartamide) silica} a
commonly used HILIC column for peptides, has been reported to exhibits
IEX interactions(5,6). Thus, salts or nonvolatile acids such as orthophosphoric
acid are recommended in the mobile phase to eliminate such effects. Peptide
isolation su
bsequently becomes more tedious with the use of a PolyHydroxyethyl
A type column.
The effect of ACN concentration on peptide retention was also examined. Opposite of RPLC, increasing the percent organic increases retention of polar compounds with HILIC stationary phases. As evident in Figure 2 , the higher the initial concentration of ACN, the better the resolution. Resolution of all peptides is possible above an 85% initial ACN concentration. Furthermore, recovery of the various peptides averaged 92% as shown in Table 1 . Recovery with the Amide-80 column was higher when compared to a column packed with an 80 pore size, end capped ODS material(3).
Reproducibility and repeatability were determined by examining both the variability between injections and the variability between columns. Ten replicates of a peptide mixture were injected on a new TSKgel Amide-80 column and a TSKgel Amide-80 column that had seen over 500 injections. As expected, the coefficient of variation for the retention time of the various peptides between injections averaged below 0.4% for both columns. The coefficient of variation for peak heights, which relates directly to efficiency, averaged 1.2% and 2.4% for the new and used columns respectively. Figure 3 provides a visual comparison of traces from a new and used column indicating that good reproducibility and long lifetimes can be achieved with this method and column type.
Conclusions:
The separation of polar compounds has long been problematic for reasons
related to retention, peak shape and reproducibility. The data shared
within in
dicates the superior performance of both the TSKgel Amide-80
column and HILIC method for the separation of peptides. The carbamoyl
stationary phase of the TSKgel Amide-80 offers a unique selectivity and
long lifetime for scientists searching for alternatives to separating
polar compounds.
References:
1. J. Dolan, Retaining Polar Compounds LC/GC Vol 5, No. 2 (June 2002)
2. T. Yoshida, Anal Chem. 69 (1997) 3038-3043
3. T. Yoshida, J. Chromatogr. A 808 (1998) 105-112
4. T. Yoshida & T. Okada, J. Chromatogr. A 840 (1999) 1-9
5. A. Alpert, J. Chromatogr. 499 (1990) 177-196
6. B.Y. Zhu et. al. J. Chromatogr. 548 (1991) 13-24
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