Confirmation That Only Minimal Levels (≤ 4 pg) of Polyethylene Glycol (PEG) Are Present in Waters Nano-LC Consumables
[ TECHNOLOGY BRIEF ]
1
GOAL
Confirmation of effectiveness of controls
during manufacturing to maintain PEG
content at minimal levels (≤ 4 pg) in
Waters™ nano-LC consumables.
BACKGROUND
Polyethylene glycol (PEG) is used in a wide
range of applications in chemistry, biology,
and medicine. However, the presence of
unwanted PEG, which can be introduced
through a variety of sources, has the
potential to impact proteomic and other
nano LC-MS separations. Verification that
manufacturing controls employed by Waters
ensure only minimal levels of PEG are
present in Waters nano-LC consumables is
an important reassurance for the users of
these products.
THE SOLUTION
Waters ACQUITY™ UPLC™ M-Class
Symmetry™ C
18 Trap Columns, 100 Å, 5 µm,
180 µm x 20 mm, 2G, V/M (p/n: 186007496)
were evaluated for the level of PEG present
post manufacturing. We tested the traps
(‘Subject Trap’) using the customized system
consisting of an ACQUITY UPLC M-Class
Waters nano columns and traps are free from meaningful
amounts of PEG.
System in conjunction with a Xevo™ G2-XS QTof Mass Spectrometer
(Figure 3). Briefly it was a modified trap-and-elute setup that allowed
downstream trapping and analysis of PEG flushed from a test subject.
The trap-and-elute setup consisted of an ACQUITY UPLC M-Class HSS T3
Column, 1.8 µm, 75 µm x 150 mm (p/n: 186007473), marked as ‘Test Column’,
and an ACQUITY UPLC M-Class Symmetry C
18 Trap Column, 100 Å, 5 µm,
180 µm x 20 mm, 2G, V/M (p/n 186007496), marked as ‘Test Trap’. A Waters
Universal NanoFlow™ Sprayer MS Source with pre-cut PicoTip™ Emitters
(p/n: 186003916) interfaced the ACQUITY UPLC M-Class System and the
Xevo G2-XS QTof MS. PEG standards, used during the method development,
and internal standards were prepared using PEG and Leucine Enkephalin
(LeuEnk) from the Q-Tof Standards Kit without Bovine (p/n: 700004768).
4 µL of the internal standard was introduced via partial loop injection from
a 5 µL loop. The acetonitrile (ACN), water, and formic acid (FA) used were
Optima™ LC-MS grade supplied by Fisher Scientific.
Figure 1. A sample MS spectra showing PEG from a contaminated LC-MS system. Note the
44 amu spacing between the peaks which is characteristic of PEG. The inset shows the
general molecular formula of PEG.
Confirmation That Only Minimal Levels (≤ 4 pg) of Polyethylene Glycol (PEG)
Are Present in Waters Nano-LC Consumables
Pádraig Kelly and Moon Chul Jung
Waters Corporation, Milford, MA, USA
[ TECHNOLOGY BRIEF ]
2
Figure 2. Calibration curve prepared using a simplified setup consisting of a µBSM
and µSM-FL. Injection volume was 4 µL.
PEG from either the mobile phases or LC
components (system PEG) could potentially
impact the results leading to false positive
results. A Symmetry C
18 Column, 100 Å, 3.5 µm,
2.1 mm x 150 mm (p/n: WAT16005), placed on
Auxiliary Solvent Manager (ASM) Line A, served
as a scrubber column to prevent system PEG
from entering the trap-and-elute setup. As the
scrubber column can become saturated with
PEG, it is important that the column is regularly
purged with acetonitrile to remove any collected
PEG (22 minutes @ 0.5 mL/min flowrate).
It is reconditioned by purging with water
(240 minutes @ 49.5 µL/min flowrate)
before reconnecting it to the test system.
Initial work using the Micro Binary Solvent
Manager (µBSM) and the Micro Sample Manager
– Fixed Loop (µSM-FL) demonstrated that
preparation of a calibration curve was possible
using PEG standards of known concentration,
see Figure 2. This method was sensitive to
PEG concentrations as low as 1 ppb from 4 µL
injections, or 4 pg.
Using this test system setup in Figures a and
3b and the method shown in Table 1, it was
possible to flush PEG from the test subject (a
trap column) and simultaneously quantify it
using the downstream trap-and-elute setup.
At the start of each run, the Subject Trap was
flushed with 10:90 water/ACN at 0.5 µL/min
(from ASM line B) to release any PEG in the trap
(Figure 3a). The highly organic flush was mixed
with water at 49.5 µL/min (from ASM line A) to
bring down the ACN concentration to below 1%,
assisting any PEG in the flush to be captured at
the Test Trap. The flush/trap process lasted for 10
minutes, which was experimentally determined
to ensure complete recovery of PEG. It was also
extremely important to accurately control the
valve switching time and the ratio of the flow
rates from two ASM lines in order to capture PEG
in the Test Trap. At the end of the flush/trap step,
the TVM valves switched the flow path to isolate
the Subject Trap and put the Test Column online
(Figure 3b). A gradient flow at 0.5 µL/min was
supplied from µBSM to start the separation.
As verification, we dosed the subject trap column
with known amounts of PEG and quantitated
the PEG using the above described test setup
Figure 3a. Test system setup during the flush/trap step. The blue lines denote the active
flow path.
Figure 3b. Test system setup during the elute and analysis step. The blue lines denote the active
flow path.
Waters Corporation
34 Maple Street
Milford, MA 01757 U.S.A.
T: 1 508 478 2000
F: 1 508 872 1990
www.waters.com
[ TECHNOLOGY BRIEF ]
Waters, The Science of What’s Possible, ACQUITY, UPLC, Symmetry, NanoFlow, PicoTip, and Xevo are
trademarks of Waters Corporation. All other trademarks are the property of their respective owners.
©2018 Waters Corporation. Produced in the U.S.A. December 2018 720006427EN TC-PDF
and the method. As the presence of system PEG
could potentially impact results, it was important
to run blank samples and correct any results for
system PEG.
The developed method was applied to test
manufactured trap columns. The results shown
in Figure 4 are the average of two runs corrected
for any system PEG.
SUMMARY
This study confirms the effectiveness of
controls during manufacturing to produce trap
columns with minimal levels of PEG (≤ 4 pg).
We developed a method to quantify the PEG
content of nano-LC trap columns using a
modified trap-and-elute setup. A key aspect
of the method is the use of a scrubber column
to reduce the amount of PEG present in the
LC system. The method developed to detect
and measure PEG present in nano-LC traps is
sensitive to 4 pg.
Figure 4. PEG content of manufactured traps in comparison to control (dosed with 400 pg PEG).
Table 1. Method for quantifying PEG on subject trap column.
µBSM
ASM line A (water)
ASM line B
(10:90 water/ACN)
TVM position
Mode
Time (min)
Flow rate
(µL/min)
Water/
ACN
Time (min)
Flow rate
(µL/min)
Time (min)
Flow rate
(µL/min)
L
R
Flush/trap
0
10
99/1
0
70
0
0
1
2
Flush/trap
3
0.5
99/1
0.5
49.5
2
0
1
2
Flush/trap
3.1
0.5
99/1
0.8
49.5
2.2
0.5
1
2
Flush/trap
10
0.5
99/1
10
49.5
10
0.5
1
2
Analysis
0
0.5
99/1
0
5
0
0.5
2
1
Analysis
0.5
0.5
99/1
0.5
5
0.5
0.5
2
1
Analysis
11
0.5
10/90
5
5
5
0.5
2
1
Analysis
18
0.5
10/90
18
5
18
0.5
2
1
Analysis
20
0.5
99/1
20
5
20
0.5
2
1
Analysis
36
0.5
99/1
36
5
36
0.5
2
1
Analysis
36.1
0.5
99/1
36.1
5
36.1
0.05
2
1
Analysis
40
0.5
99/1
40
5
40
0.05
2
1
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