Supplementary Material
Membrane-based Continuous Remover of Trifluoroacetic Acid in Mobile
Phase for LC-ESI-MS Analysis of Small Molecules and Proteins
Zhigui Zhou, Jialing Zhang, Jiawei Xing, Yu Bai, Yiping Liao, and Huwei Liu*
Beijing National Laboratory for Molecular Sciences, the Key Laboratory of Bioorganic
Chemistry and Molecular Engineering of Ministry of Education, Institute of Analytical
Chemistry, College of Chemistry and Molecular Engineering, Peking University, Beijing
100871, China
Correspondence to: Huwei Liu, Institute of Analytical Chemistry, College of Chemistry and
Molecular Engineering, Peking University, Beijing 100871, China, fax: +86-10-62751708,
e-mail: hwliu@pku.edu.cn.
Experimental
Chemicals and Reagents
Ribonuclease A (RNase A), cytochrome c (CYC, equine heart), and lysozyme (LYS) were
obtained from Merck (NJ, U.S.A.), and myoglobin (MYG, equine) was from Sigma-Aldrich
(MO,
U.S.A.).
N--benzoyl-L-arginine
ethyl
ester
(99%,
BAEE)
and
N--benzoyl-L-arginine (99%, BA) were purchased from Alfa Aesar (MA, U.S.A.).
Trifluoroacetic acid (TFA) was from Acros Organics (NJ, U.S.A.). HPLC grade acetonitrile
(ACN) and formic acid (FA) were purchased from Dikma Technologies Inc. (CA, U.S.A.).
Purified water was provided by Wahaha Group Co., Ltd. (Hangzhou, China). All the reagents
were used without further purification.
TFA Remover fabrication
As shown in Figure S1, the TFA remover was constructed with two electrodes, two
polycarbornate (PC) slices with a channel (40 mm long, 1 mm wide, 1 mm deep) in the
middle to deliver running buffer, a bipolar membrane, a anion exchange membrane, and a
poly(tetrafluoroethylene) (PTFE) film with a micro-channel (40 mm long, 1 mm wide, 50 μm
deep) in the middle to deliver LC mobile phase. The bipolar membrane with sulfonic and
quaternary ammonium functional groups as ion exchangers was purchased from Beijing
Tingrun Membrane Technology Development Co., Ltd. (Beijing, China), and the anion
exchange membrane was from Unisplendour Co., Ltd. (Beijing, China). The channels in the
PC slices and PTFE film were mechanically fabricated through the slices and film. The PTFE
film was sandwiched between the bipolar membrane, anion exchange membrane, and PC
slices as shown in Figure S1. Two additional channels with the same size (400 μm wide, 400
μm deep, 12 mm long) were mechanically fabricated on both ends of the PC slice at the side
of the anion exchange membrane. Two corresponding channels on the anion exchange
membrane were made by mildly pressing the membrane into the channels with a capillary to
leave two prints. Two PEEK capillaries (360 μm o.d, 100 μm i.d.) were embedded and fixed
into the two channels by epoxy resin glue to deliver mobile phase through the micro-channel.
Two electrodes were placed at the outer sides of the PC slices, and connected with the power
supply. All these units were clamped between two aluminum plates and fixed by four screws.
Holes were drilled in the electrodes and aluminum plates to deliver running buffer (20 mM
NH4HCO3) through the channels in the PC slices for TFA removal.
Instrument Setup of LC-TFA remover-MS System
All LC experiments were performed on an Agilent 1100 series HPLC system (Agilent
Technologies, CA, U.S.A.). As shown in Figure 1(a), the inlet of the TFA remover was
coupled with LC column via a commercial T-connection for mobile phase splitting if
necessary. The splitting ratio was controlled by the length and inner diameter of the tubing
that connected the T-connection and the waste bottle. The outlet tubing of the remover was
directly connected to the ESI source of MS.
Sample Preparation
The stock solution of BAEE and BA mixture was prepared in 50 mM NH4HCO3 at the
concentration of 2 mM each. Working solutions (0.2, 0.5, 1, 2, 5, 10, 20, 50, 100, and 200 μM)
were prepared by diluting the stock solution with 50 mM NH4HCO3 to the required
concentration. Proteins (RNase A, CYC, LYS, and MYG) were respectively dissolved in 50
mM NH4HCO3 at the concentration of 1 mg/mL and stored at -20 °C. The stock solution of
protein mixture (0.5 mg/mL each) was prepared by mixing the individual protein solutions in
50 mM NH4HCO3. The stock solution was diluted with 50 mM NH4HCO3 to obtain the
working solutions (0.2, 0.5, 1, 2, 5, 10, 20, 50, 100, 200, and 500 μg/mL). All stock solutions
were stored under refrigerated conditions (4°C), and all solutions were filtered through 0.22
μm cellulose membrane before injection.
LC and MS conditions
An Agilent HC-C8 column (150 mm × 2.1 mm i.d., 5 μm particle size, Agilent Technologies,
CA, U.S.A.) was utilized for the separation of BAEE and BA. The mobile phase was water
and ACN with TFA as modifier. UV at 254 nm was used for the detection. Proteins were
analyzed on a Zorbax 300SB-C18 column (150 mm × 2.1 mm i.d., 5 μm particle size, Agilent
Technologies, CA, U.S.A.). The mobile phase consisted of water (containing 0.1% FA or TFA,
solvent A) and ACN (containing 0.1% FA or TFA, solvent B), and the flow rate was 0.2
mL/min. The optimized gradient was: 0-25 min 30%-65% B, 25-35 min 65% B, 35-50 min
30% B. For UV detection, the wavelength was set to 210 nm.
All MS experiments were performed on an Agilent XCT ion trap mass spectrometer (Agilent
Technologies, CA, U.S.A.). Positive mode was used for all experiments, and the parameters
were set as follows: nebulizer 35 psig, dry gas temperature 325 °C, and dry gas flow 8 L/min.
The MS data were acquired at the rate of 1.03 spectra/s in the mass range of m/z 100–600 for
BAEE and BA and m/z 100–2200 for proteins. MS data was processed using DataAnalysis
3.3 (Bruker Daltonik GmbH) for Agilent ion trap MS. The extracted ion chromatograms
(EICs, ±0.5 m/z expansion) of BAEE (m/z 307.4), BA (m/z 279.3), RNase A (m/z 1053.5),
CYC (m/z 816.3), LYS (m/z 1101.3), and MYG (m/z 848.6) were extracted and integrated,
respectively.
Figure S1. Expanded view of the designed TFA Remover.
Figure S2. Total conductivities and pH values of the tested mobile phases flowing out of the
remover under different flow rates and voltages: (a) and (b) water (containing 0.5% TFA); (c)
and (d) ACN/water (50/50, v/v, containing 0.5% TFA); (e) and (f) water (containing 0.1%
TFA); (g) and (h) ACN/water (50/50, v/v, containing 0.1% TFA)
Figure S3. Total conductivities of water (a) and ACN/water (50/50, v/v, b) flowing out of the
remover under different flow rates and voltages
Figure S4. (a) (b) Typical chromatograms of BAEE and BA without (a) and with (b) TFA as
modifier in the mobile phase; (c) typical chromatogram of BAEE and BA using LC-TFA
remover-UV system; (d) combined EICs of BAEE and BA obtained by LC-TFA remover-MS
system
Figure S5. Typical chromatograms of four proteins using FA (a) and TFA (b) as modifier in
the mobile phase
Figure S6. Combined EICs of four proteins using LC-TFA Remover-MS system under
different remover voltages: (a) 5 V; (b) 8 V; (c) 11 V
Table S1. Peak Areas of BAEE and BA Using UV Detector With and Without TFA Removal
(n = 3)
Without TFA Removal
With TFA Removal
BA
2561
3870
BAEE
2524
2139
Table S2. Quantitative Analysis Results of BAEE and BA Using LC-TFA remover–MS
System (n=3)*
BA
Concentration
BAEE
μM
Average area
RSD%
Average area
RSD%
0.2
91850736
21.9
96676626
9.8
0.5
103516183
2.7
236306144
1.5
1
153746374
1.7
336916671
1.1
2
300060670
1.0
457646998
0.2
5
669831969
0.1
1128752728
1.4
10
1284170156
2.9
2048397682
0.8
20
2296288389
1.2
3452519634
1.4
50
5159487972
0.5
8639170653
0.3
100
8434985861
0.4
15457365482
0.9
200
13258694913
1.5
26353756576
1.4
*The injection volume was 2 μL. The voltage of TFA remover was 10.5 V. The extracted ion
chromatograms (EICs, ±0.5 m/z expansion) of BAEE (m/z 307.4) and BA (m/z 279.3) were
extracted and integrated.
Table S3. Quantitative Analysis Results of Four Proteins Using LC-TFA remover–MS
System (n=3) *
Concentration
RNase A
CYC
μg/mL
Average area
RSD%
Average area
RSD%
500
133470469
1.6
5346055936
1.5
200
58162085
6.3
2390222152
2.3
100
27153302
2.0
1085214485
1.2
50
13582525
7.9
554323892
0.8
10
4047666
6.3
75835380
0.8
5
2739096
17.2
33554511
7.1
(Continued)
LYS
Concentration
MYG
μg/mL
Average area
RSD%
Average area
RSD%
500
45288935
1.0
7209702274
0.4
200
16100213
1.0
2971252906
0.8
100
8694860
3.5
1531549823
1.0
50
5383699
3.4
760094700
0.7
10
1993456
1.3
153376556
2.1
5
781522 (LOD)
-
82652611
4.0
1
Not detected
-
17665235
4.0
0.5
Not detected
-
6581537
1.5
*The injection volume was 5 μL. The voltage of TFA remover was 6 V. The extracted ion
chromatograms (EICs, ±0.5 m/z expansion) of RNase A (m/z 1053.5), CYC (m/z 816.3), LYS
(m/z 1101.3), and MYG (m/z 848.6) were extracted and integrated.