Confirmation That Only Minimal Levels (≤ 4 pg) of Polyethylene Glycol (PEG) Are Present in Waters Nano-LC Consumables

[ TECHNOLOGY BRIEF ]

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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 ]

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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