ACQUITY UPLC Glycoprotein BEH Amide, 300Å, 1.7 μm Columns and Glycoprotein Performance Test Standard

CONT ENTS

I.   INT RODUCTION

II.  GET TING START ED
 

a. eCord™ Installation

b. Column Connectors

c. Column Installation

d. Column Equilibration

e. Initial Column Efficiency Determination 

f.  Conditioning of Previously Unused Columns  

  and LC System Considerations

g. Useful Functional Tests for Benchmarking 

  a New Column: Glycoprotein Performance  

  Test Standard

III.  COLUMN USE
 

a. Sample Preparation

b. Operating pH Limit

c. Solvents

d. Pressure

e. Temperature 

IV. T ROUBLESHOOTING

V. 

COLUMN CLEANING, REGENERATION, 

AND STORAGE

a. Cleaning and Regeneration

b. Storage

VI. INT RODUCING 

eCORD INT ELLIGENT 

CHIP T ECHNOLOGY

a. Introduction

b. Installation

c. Manufacturing Information

d. Column Use Information

VII.  CAUTIONARY NOT E

VIII.  ORDERING INFORMATION

ACQUITY UPLC Glycoprotein BEH Amide, 300Å, 1.7 μm Columns 

and Glycoprotein Performance Test Standard

I. INT RODUCTION
Thank you for choosing a Waters ACQUITY UPLC® Glycoprotein 
BEH Amide, 300Å, 1.7 µm Column that has been purposefully 
designed for intact glycoprotein profiling, subunit fragment 
analysis, and glycopeptide mapping using hydrophilic interaction 
chromatography (HILIC). These orthogonal analytical techniques 
provide additional means of characterizing protein glycosylation 
by assisting in glycan identification or in elucidating sites of 
glycan occupancy as documented in Waters Application Notes 
Entitled: “Developing High Resolution HILIC Separations of Intact 
Glycosylated Proteins Using a Wide-Pore Amide-Bonded Stationary 
Phase” (720005380EN), “Mapping IgG Subunit Glycoforms Using 
HILIC and a Wide-Pore Amide Stationary Phase” (720005385EN), 
and “HILIC Glycopeptide Mapping with a Wide-Pore Amide 
Stationary Phase” (720005409EN).

The high resolving power of this amide-based, UPLC® Column is 
due in part to the small particle size (1.7 μm) of the fully-porous 
packing material when used on an appropriately configured UPLC 
System. Outstanding chemical and mechanical stability of the 
column, that translates into long column life, are the result of 
using Waters ethylene bridged hybrid (BEH) particle coupled to 
innovative Amide bonding technology.

See Pages

10–13 for

Tips & Tricks

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To help ensure column-to-column consistency, each batch of 
Waters ACQUITY UPLC Glycoprotein BEH Amide, 300Å, 1.7 µm 
material is quality control tested with Waters Glycoprotein 
Performance Test Standard (p/n 186008010). This same 
glycoprotein standard (i.e, ribonuclease glycoforms) is included 
with each shipped column to assist users in obtaining optimal 
performance with a new column, and monitoring column and 
system performance over time.

The ACQUITY UPLC Glycoprotein BEH Amide, 300Å Column 
is based on the 1.7 µm particles that are characteristic of 
UltraPerformance LC®. The small particle size packing reduces 
dispersion and band broadening so that improved resolution, 
sensitivity, and speed are obtained in glycan separations. It is 
commonly expected that very high system backpressures will be 
observed with such small particles. The high pressure capability 
of the ACQUITY UPLC, ACQUITY UPLC H-Class, and ACQUITY 
UPLC H-Class Bio Systems is required for UPLC glycan analysis. 
With HILIC, back pressure increases with the increasing water 
content during the gradient. If an aqueous wash is chosen before 
re-equilibration, it might be necessary to use a lowered flow rate 
to prevent excessive back pressure.

Note: ACQUITY UPLC Glycoprotein BEH Amide, 300Å, 1.7 μm Columns are 
optimally designed for use with the ACQUITY UPLC, ACQUITY UPLC H-Class, 
and ACQUITY UPLC H-Class Bio Systems. The expected separations will not be 
obtained on a conventional HPLC system because of excessive dispersion and 
pressure limitations. 

II. GET TING START ED
Each ACQUITY UPLC Glycoprotein BEH Amide, 300Å, 1.7 μm 
Column has a Certificate of Analysis and a Performance Test 
Chromatogram. The Certificate of Analysis is specific to each 
batch of packing material and includes the batch number and 
analyses of the physical and chemical properties of the particle. 
Particle size and pore structure are analyzed prior to bonding. 
The carbon and nitrogen content of the bonding are measured 
to insure consistent coverage. The selectivity of each batch is 
also assessed with the chromatographic separation of Waters 
Glycoprotein Performance Test Standard that consists of a 
mixture of Ribonuclease A and Ribonuclease B glycoforms 
(p/n 186008010). The retention times and resolution of 
selected components are used as the quality control test for each 
batch of packing material. The Performance Test Chromatogram 
is specific to each individual column and contains the following 

information: batch number, column serial number, backpressure, 
USP plate count, reduced plate height (RPH), USP tailing factor, 
retention factor (k’), peak width, and chromatographic conditions. 
These data can be found on the eCord supplied with each column 
and should be stored for future reference. 

a. eCord Installation
The eCord button should be attached to the side of the column 
heater module. The eCord button is magnetized and does not 
require specific orientation.

b. Column Connectors
The ACQUITY UPLC, ACQUITY UPLC H-Class, and ACQUITY UPLC 
H-Class Bio Systems utilize tubing and gold plated compression 
screws which have been designed to meet stringent tolerance 
levels and to minimize system dispersion.

For the ACQUITY UPLC System, columns should be attached to 
the injector with a column stabilizer, of which there are 4 types:



 205000291  50 or 100 mm column



 205000365  150 mm column



 205000489  HTCH 50 or 100 mm



 205000494  HTCH 150 mm

The first two parts are for the original heater and differ in the 
tubing arrangement to allow 150 mm columns to be used with 
a VanGuard™ pre-column or in-line filter while stabilizing the 
solvent temperature. The second two parts are for the newer 
high temperature column heater (HTCH). Optimized column inlet 
tubing is supplied with the ACQUITY UPLC System. The inject 
valve end of the tubing is clearly marked with a blue shrink tube 
marker. Insert the opposite end of the tubing into the ACQUITY 
UPLC Column and tighten the compression fitting using two 
5/16-inch wrenches (or finger tighten the knurled nut).

If this column will be used on an ACQUITY UPLC H-Class 
or ACQUITY UPLC H-Class Bio System, simply connect the 
column to the active preheater supplied on the system using the 
gold fingertight fittings. There is only one configuration of 
column stabilizer on the ACQUITY UPLC H-Class and 
ACQUITY UPLC H-Class Bio Systems.

For more information on fittings and connective tubing, please 
refer to the relevant sections of the operator’s guides for the 
ACQUITY UPLC, ACQUITY UPLC H-Class, and ACQUITY UPLC 
H-Class Bio Systems.

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ACQUITY UPLC Glycoprotein BEH Amide, 300Å, 1.7 μm Columns and Standard

c. Column Installation
Note: The flow rates given in the procedure below are for a typical 1.7 μm 
packing in a 2.1 mm I.D. column. 

1.   Purge the solvent delivery system of any buffer-containing 

or water-immiscible mobile phases and connect the inlet 
end of the column to the injector outlet. An arrow on the 
column identification label indicates the correct direction 
of solvent flow.

2.   Flush the column with 100% organic mobile phase 

(acetonitrile) by setting the pump flow to 0.1 mL/min and 
increase the flow to 0.25 mL/min over 3 minutes. Increase 
the aqueous phase to 90% over 10 minutes. Note the 
backpressure. Decrease aqueous phase to starting 
conditions (15% aqueous in the test chromatogram).

3.   When the mobile phase is flowing freely from the column 

outlet, stop the flow and attach the column outlet to the 
detector. This prevents entry of air into the detection 
system and gives more rapid baseline equilibration.

4.   Gradually increase the flow rate from 0.25 to 0.5 mL/min 

over 3 minutes.

5.   Once a stable backpressure and baseline have been 

achieved, proceed to the next section. 

d. Column Equilibration 
Glycoprotein columns are shipped in 100% acetonitrile. It is 
important to ensure mobile phase compatibility before changing 
to a different mobile-phase system. Equilibrate the column with a 
minimum of 10 column volumes of the mobile phase to be used 
(refer to Table 1 for column volumes). 

Table 1. Empty column volumes in mL 
(multiply by 10 for flush solvent volumes) 

To avoid precipitating mobile-phase buffers on your column 
or in your system, flush the column with five column volumes 
of a water/organic solvent mixture using the same or higher 
acetonitrile content as in the desired buffered mobile phase. 
For example, flush the column and UPLC system with 50% 
acetonitrile in water prior to introducing 50% acetonitrile/50% 
buffered mobile phase.

Column equilibration may be judged initially by stable pressure 
and by a stable detector baseline. For a specific application, it is, 
however, necessary to test the required duration of equilibration. 
The criteria for adequate equilibration include reproducibility of 
retention time for major and minor peaks, resolution for critical 
pairs, and consistent baseline characteristics.

Note: Low concentration mobile-phase additives, particularly those with minimal 
buffering capacity, may require extended equilibration and re-equilibration 
between gradient analyses.

e. Initial Column Efficiency Determination 
1.   Perform an efficiency test on the column before using it 

in the desired application. Waters recommends using the 
solute mixture and conditions described in the “Performance 
Test Chromatogram” to test the column upon receipt. These 
conditions can be found on the eCord attached to the column.

2.   Measure the retention of the test compounds and the number 

of theoretical plates (N). 

3.   Repeat the test at predetermined intervals to track column 

performance over time. Slight variations may be obtained 
on two different UPLC Systems due to the quality of the 
connections, operating environment, system electronics, 
reagent quality, condition of column, and operator technique.

Column length

Volume in mL (2.1 mm I.D.)

50

0.17

100

0.35

150

0.52

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ACQUITY UPLC Glycoprotein BEH Amide, 300Å, 1.7 μm Columns and Standard

f. Conditioning of Previously Unused Columns 
and LC System Considerations

New (previously unused) ACQUITY UPLC Glycoprotein BEH 

Amide, 300Å, 1.7 µm Columns should be conditioned, before 
actual test sample analyses. This can be accomplished via 
sequential injections of a representative sample until a stable 
chromatographic profile is achieved. Alternatively two 40 µg 
injections of Glycoprotein Performance Test Standard (10 µL 
at 4 mg/mL in 0.1% TFA, 80% ACN) could be used. 

EU 

0.00

5.00

10.00

15.00

20.00

25.00

30.00

35.00

40.00

45.00

50.00

6

EU 

0.00

5.00

10.00

15.00

20.00

25.00

30.00

35.00

40.00

45.00

50.00

8

10

12

14

16

18

20

22 min.

4

6

8

10

12

14

16

18

20

22 min.

4

Chromatography after a single, 40 µg

protein column conditioning injection

Chromatography after two, 40 µg

protein column conditioning injections

 
Figure 1. Conditioning of a previously unused ACQUITY UPLC Glycoprotein BEH 
Amide, 300Å, 1.7 μm Column. Top: Separation of Glycoprotein Performance Test 
Standard after a single column conditioning with 40 μgs of same Glycoprotein 
Performance Test Standard. Bottom: Overlay of two sequential chromatograms 
of the Glycoprotein Performance Test Standard after two, 40 μg injections 
(i.e., 80 μgs total protein) of same Glycoprotein Performance Test Standard.

It should also be noted that even if the Glycoprotein BEH 
Amide column is to be used with an alternative mobile phase 
system, such as ammonium formate, it can still prove useful to 
first condition it with a gradient and mobile phases containing 
0.1% TFA. TFA can be effective in cleaning a column’s station-
ary phase by both neutralizing and ion pairing contaminants 
that in their ionic form might otherwise strongly adsorb to a 
HILIC stationary phase. 

In addition, LC systems might require passivation in order to 
achieve optimal HILIC separations. It is advised to use metallic 
flow paths where ever possible, including post column tubing 
that extends to optical detectors and/or the divert valve of a 
mass spectrometer. Stainless steel and MP35N alloy materials 

have been found to show better compatibility with large 
molecule HILIC than PEEK.

g. Useful Functional Tests for Benchmarking a New Column: 
Glycoprotein Performance Test Standard
We suggest use of Waters Glycoprotein Performance Test 
Stardard (p/n 186008010) to benchmark your new column and 
monitor its performance during use.

Stability: Please store the standard in the original packaging at 
-20 °C upon arrival for long-term storage before solubilization. 
Once solubilized, the standard can be aliquoted and stored frozen 
for up to 3 months or stored at 4-10 °C for no more than a week, 
but avoid any freeze/thaw that could lead to degradation.

Recommended Reconstitution: Reconstitute the contents of 
each vial with 25 μL of 0.1% TFA in 80% ACN. For conditioning 
a previously unused 2.1 mm ID ACQUITY UPLC Glycoprotein 
BEH Amide, 300Å, 1.7 μm Column, perform two sequential 
gradient separations of the standard injected at a volume of 
10 μL. For benchmarking and monitoring the performance of 
a 2.1 mm ID ACQUITY UPLC Glycoprotein BEH Amide, 300Å, 
1.7 μm Column, perform gradient separations of the standard 
injected at a volume of 0.5 μL.

Generic Chromatographic Method
The conditions shown below are for use on an ACQUITY UPLC 
H-Class or ACQUITY UPLC H-Class Bio System, wherein 70/30 
acetonitrile/water is used for the purge and wash solvents. 
Note that the ACQUITY UPLC H-Class and ACQUITY UPLC 
H-Class Bio Systems do not have a strong and weak needle 
wash. Instead, they have one purge and one wash solvent, both 
of which should be in 70/30 acetonitrile/water. 

It might be necessary to avoid high organic diluents for some 
samples due to the propensity for proteins to precipitate 
under ambient conditions. A 2.1 mm ID column can typically 
accommodate up to a 1.2 µL aqueous injection before 
chromatographic performance is negatively affected. 

High ACN diluents can sometimes be used in intact protein 
HILIC, but care must be taken to enhance the solubility of the 
protein sample through either the use of TFA ion pairing at 
concentrations between 0.2 to 1%, the combined application of 
TFA and hexafluoroisopropanol (HFIP), or by use of co-solvents, 
such as dimethylsulfoxide (DMSO).

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ACQUITY UPLC Glycoprotein BEH Amide, 300Å, 1.7 μm Columns and Standard

m/z

1000

1500

2000

2500

3000

3500

%

0

100

%

0

100

%

0

100

%

0

100

%

0

100

%

0

100

aglycosylated

+Man5

+Man6

+Man7

+Man8

+Man9

6+

7+

8+

9+

5+

KETAA AKFER QHMDSSTSAA

CNQMMKSRNL

TKDRC KPVNT

AVCSQ KNVAC KNGQT

NCYQSYSTMS ITD CR ETGSS KYPNC AYKTT QANKH
IIVA C EGNPYVPVHF DASV

Disulfides

Injection volume:   

1.0 µL

Column:   

ACQUITY UPLC Glycoprotein

BEH Amide, 300Å, 1.7 μm

2.1 x 150 mm (p/n 186007963)

Eluent A:  

0.1% (v/v) TFA, H2O

Eluent B:  

0.1% (v/v) TFA, ACN

Wash and purge solvents:  Acetonitrile/HPLC grade water, 
   

(70/30 

v/v)

Temperature: 

45 °C

Native Fluorescence
Detection: 

Fluorescence: λex = 280 nm, 

λem = 320 nm

UV Detection: 

214 / 280 nm, 2 Hz

Flow: 

  0.2 

mL/min

Gradient: 

Time 

%A 

%B 

Curve

0.0  15.0  85.0 6

0.5  15.0  85.0 6

1.0  33.0  67.0 6

21.0 40.0  60.0 6

22.0 100.0  0.0  6

24.0 100.0  0.0  6

25.0 15.0  85.0 6

35.0 15.0  85.0 6

EU

0.00

20.00

40.00

60.00

80.00

100.00

120.00

140.00

160.00

5

10

15

20 min

1 

2 

3 

4 

5 

6 

Glycoprotein 

Performance 

Test Standard 

Peak 

Species 

Rs 

1 

RNase A 

- 

2 

RNase B (+Man 5)  21.2 

3 

RNase B (+Man 6) 

3.5 

4 

RNase B (+Man 7) 

2.7 

5 

RNase B (+Man 8) 

2.6 

6 

RNase B (+Man 9) 

3.1 

 
Figure 2. Separation of the Glycoprotein Performance Test Standard 
(RNase A + RNase B glycoforms) using an ACQUITY UPLC Glycoprotein BEH Amide, 
300Å, 1.7 μm, 2.1 x 150 mm Column. Fluorescence detection at Ex 280 nm and Em 
320 nm and a column temperature of 45 ˚C were employed in this example.

Additive/Buffer

pKa

Buffer 

range (±1 

pH unit)

Volatility

Used for 

Mass 

Spec

Comments

Trifluoroacetic acid

0.23

–

Volatile

Yes

TFA ion pairing is often essential in large molecule HILIC separations as it improves the 
solubility of proteins in high ACN mobile phase and shields the proteinaceous residues from 
interacting with the amide stationary phase.  TFA ion pairing therefore improves peak shape, 
selectivity and resolution of the hydrophilic modifications on proteins and peptides 
(Waters Application Note 720005380EN)

Formic acid

3.75

–

Volatile

Yes

Maximum buffering obtained when used with ammonium formate salt. Used in 0.1-1.0% range.

Table 2. Buffer Recommendations for Using ACQUITY UPLC Glycan BEH Amide, 300Å, 1.7 μm Columns

Species

MAverage (Da)

Aglycosylated (4 disulfides)

13682.3

RNase B (4 disulfides), Man5

14899.4

RNase B (4 disulfides), Man6

15061.6

RNase B (4 disulfides), Man7

15223.7

RNase B (4 disulfides), Man8

15385.8

RNase B (4 disulfides), Man9

15548.0

Figure 3. Sequence Information and MS Data for the Glycoprotein Performance 
Test Standard.

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ACQUITY UPLC Glycoprotein BEH Amide, 300Å, 1.7 μm Columns and Standard

III. COLUMN USE
To ensure the continued high performance of the ACQUITY UPLC 
Glycoprotein BEH Amide, 300Å, 1.7 µm Columns, observe the 
following guidelines:

a. Sample Preparation 
1.  Sample impurities often contribute to column contamination. 

Samples should be free of particles before injection into 
the system. 

2.  If the sample is not dissolved in the mobile phase or 

solvent combinations specified in this manual, ensure 
that the sample, solvent, and mobile phases are miscible 
in order to avoid sample and/or buffer precipitation. It is 
good practice to remove any precipitate or particulates by 
centrifugation at >10,000 rpm for more than 2 minutes.

b. Operating pH Limits
The recommended operating pH range for the ACQUITY UPLC 
Glycoprotein BEH Amide, 300Å, 1.7 µm Column is 2 to 10. A 
listing of commonly used buffers and additives is given in Table 
2. Additionally, the column lifetime will vary depending on the 
operating temperature as well as the type and concentration of 
buffer used.

c. Solvents 
To maintain maximum column performance, use high quality 
chromatography grade solvents. If filtering, Acrodisc® filters 
are recommended. Solvents containing suspended particulate 
materials can damage the fluidic components of the UPLC 
System and will generally clog the inlet distribution frit of 
the column. This will result in higher operating pressure and 
poor performance. 

d. Pressure 
The ACQUITY UPLC Glycoprotein BEH Amide, 300Å, 1.7 µm 
Columns will have greatly increased backpressure when 
operated in 90-100% aqueous mobile phases. When employing 
long column lengths or investigating high flow rates, it might 
be necessary to be mindful of this increase in pressure and to 
potentially reduce flow rates during the aqueous regeneration 
step of the HILIC separation. ACQUITY UPLC Glycoprotein BEH 
Amide, 300Å, 1.7 µm Columns can tolerate pressures of up to 
15,000 psi (1034 bar or 103 Mpa).

Note: Working at the extremes of pressure, pH and/or temperature will result in 
shorter column lifetimes.

e. Temperature 
Temperatures between 20 ˚C – 90 °C are recommended for 
operating ACQUITY UPLC Glycoprotein BEH Amide, 300Å, 
1.7 µm Columns in order to enhance selectivity, lower solvent 
viscosity, and increase mass transfer rates. However, higher 
temperature will have a negative effect on lifetime that will vary 
depending on the pH and buffer conditions used. 

IV. T ROUBLESHOOTING
The first step in systematic troubleshooting is comparing 
the column, in its current state, to the column when it was 
functioning properly. The method suggested in Section II for 
measuring plate count is an essential first step. This technique 
detects physical changes to the packed bed and chemical 
changes in the bonded-phase surface. The functional test 
with the ribonuclease A and B glycoforms in the Glycoprotein 
Performance Test Standard may reveal more subtle changes in 
surface chemistry that affect the application.

There are several common symptoms of change in the column.

1.   An increase in pressure is often associated with lost 

performance in the application. The first step in diagnosis 
is to ensure that the elevated pressure resides in the 
column rather than somewhere else in the system. This 
is determined by measuring pressure with and without 
the column attached to the instrument. If the system is 
occluded, the blockage should be identified and removed. 
If the pressure increase originates from the column, it 
is helpful to know whether the problem was associated 
with a single injection or whether it occurred over a 
series of injections. If the pressure gradually built up, 
it is likely that the column can be cleaned as described 
below (Section V). For future stability, it may be useful to 
incorporate a stronger regeneration step in the method. 
If a single sample caused the pressure increase, it likely 
reflects particulates or insoluble components. Cleaning 
is still an option, but using the more aggressive methods. 
The sudden pressure increase suggests that the user 
should consider some sample preparation, such as high 
speed centrifugation.

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ACQUITY UPLC Glycoprotein BEH Amide, 300Å, 1.7 μm Columns and Standard

2.   Loss of retention can reflect a change in the column 

surface chemistry. Before proceeding with diagnostic or 
corrective measures, check that the mobile phases have 
been correctly prepared and the correct method has been 
selected. Then repeat the plate count test and a separation 
of the Glycoprotein Performance Test Standard. If both the 
plate count and glycoprotein test show loss of retention, it 
is likely that a significant fraction of the bonded phase has 
been lost, and the column will require replacement. If the 
changes are small and reflected only for some glycans, 
one of the cleaning procedures may be effective.

3.   Change in peak shape, resolution, or relative retention 

of peaks. Follow the same steps as for loss of retention 
(Section II).

4.   Carryover and memory effects are defined as the appearance 

of the constituents of one sample in the next gradient 
analysis. First determine whether the column or the system 
is the source of carryover. Define a gradient method that 
includes an “internal gradient”. That is, the analytical 
gradient is repeated within a single method. If the peaks 
appear in both gradients, at the same time after start, the 
carryover came from the column in what is often described 
as a “memory effect”. If the peaks only appear when an 
injection is made, they likely originated from adsorption 
to some system component. In that case follow the 
instrument manufacturer’s recommendations. Memory 
effects as a source of carryover may be reduced or 
eliminated in several ways. First, raising the temperature 
of the separation reduces the possibility of non-specific 
adsorption. Second, memory effects may be more pronounced 
with steep gradients. Keep the gradient slope at 1% per 
column volume or less. Finally, apparent memory effects 
may actually reflect the solubility of the sample in the 
mobile phase. Reducing the amount injected may eliminate 
the effect.

Note: Useful general information on column troubleshooting problems may be 
found in HPLC Columns Theory, Technology and Practice, U.D. Neue, (Wiley-VCH, 
1997), the Waters HPLC Troubleshooting Guide (Literature code # 720000181EN), 
or visit www.waters.com.

V. COLUMN CLEANING, REGENERATION, 
AND STORAGE 

a. Cleaning and Regeneration
Changes in peak shape, peak splitting, shoulders on the peak, 
shifts in retention, change in resolution, carryover, ghost peaks, 
or increasing backpressure may indicate contamination of the 
column. Choose a cleaning option that may be expected to 
dissolve the suspected contaminant.

1.   All cleaning procedures will be more effective at higher 

temperatures. It is reasonable to conduct cleaning at 70 °C.

2.   It may be useful to conduct cleaning procedures at one-

half the flow rate typically used with that column. In this 
way, the possibility of high pressure events is reduced.

3.  The first and simplest cleaning procedure is to run a series 

of gradients from 0–100% water. Be sure to reduce the 
flow rate for gradients with higher than 75% aqueous 
content. Columns of 150 mm length should be operated 
at 250 µL per minute or less during washes. The gradients 
can be as short as 5 column volumes and 3–5 repetitions 
may be effective. 

4.  Regeneration steps and flushing procedures using 

100% aqueous mobile phase can help to maintain the 
optimal peak shape and selectivity of a HILIC separa-
tion. Additionally, an analyst can perform gradients 
with mobile phases containing 0.1% TFA as a means to 
maintain or recover the performance of a Glycoprotein 
BEH Amide column. TFA can be effective in cleaning 
a column’s stationary phase by both neutralizing and 
ion pairing contaminants that in their ionic form might 
otherwise strongly adsorb to a HILIC stationary phase.

5.   Several different cleaning solutions may be injected to 

strip strongly adsorbed material or particulates from the 
column. Make the largest injection possible with the system 
configuration. With such strong cleaning solutions, it is best to 
disconnect the detector from the column and to direct the flow 
to waste.

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ACQUITY UPLC Glycoprotein BEH Amide, 300Å, 1.7 μm Columns and Standard

At the time of manufacture, tracking and quality control 
information will be downloaded to the eCord. This includes the 
conditions and results for the Performance Test Chromatogram. 
Storing this information on the chip will eliminate the need for a 
paper Certificate of Analysis. Once the user installs the column, 
the software will automatically download key parameters into a 
column history file stored on the chip. In this manual, we explain 
how the eCord will provide a solution for easily tracking 
the history of the column, reduce the frustration of paperwork 
trails, and give customers the reassurance that a well performing 
column is installed onto their instruments.

b. Installation
Install the column into the column heater. Plug the eCord 
into the side of the column heater. Once the eCord is inserted 
into the column heater, the identification and overall column 
usage information will be available in the ACQUITY UPLC 
Console, allowing the user to access column information on 
their desktop.

eCord Fob

 
Figure 5. Installing the eCord Intelligent Chip.

c. Manufacturing Information
The eCord chip provides the user with QC test conditions and results on 
the column run by the manufacturer. The information includes mobile 
phases, running conditions and analytes used to test the columns. In 
addition, the QC results and acceptance is placed onto the column.

6.   Flow reversal or backflushing is often suggested as part 

of a cleaning procedure. This should be reserved as a last 
resort. It may further damage the column or provide a 
short-lived improvement in performance.

b. Storage
For periods longer than four days at room temperature, store 
the column in 100% acetonitrile. Immediately after use with 
elevated temperatures and/or at pH extremes, store in 100% 
acetonitrile for the best column lifetime. Do not store columns 
in highly aqueous (<50 % organic) mobile phases. If the mobile 
phase contained a buffer salt, flush the column with 10 column 
volumes of HPLC grade water (see Table 1 for common column 
volumes) and replace with 100% acetonitrile for storage. Failure 
to perform this intermediate step could result in precipitation of 
the buffer salt in the column or system when 100% acetonitrile 
is introduced. Completely seal the column to avoid evaporation 
and drying out the bed. 

VI. INT RODUCING eCORD INT ELLIGENT 
CHIP T ECHNOLOGY

a. Introduction
The eCord intelligent chip provides the history of a column’s 
performance throughout its lifetime. The eCord is permanently 
attached to the column to assure that the column’s performance 
history is maintained in the event that the column is moved 
from one instrument to another.

Figure 4. eCord Intelligent Chip. 

8

[ CARE AND USE MANUAL ]

ACQUITY UPLC Glycoprotein BEH Amide, 300Å, 1.7 μm Columns and Standard

d. Column Use Information
The eCord chip provides the customer with column use data. The top 
of the screen identifies the column including chemistry type, column 
dimensions and serial number. The overall column usage information 
includes: total number of samples, total number of injections, total 
sample sets, date of first injection, date of last injection, maximum 
pressure, and temperature. The information also details the column 
history by sample set including: date started, sample set name, user 
name, system name, number of injections in the sample set, number of 
samples in the sample set, maximum pressure, and temperature in the 
sample set and if the column met basic system suitability requirements.

VII. CAUTIONARY  NOT E
Depending on users application, these products may be classified 
as hazardous following their use and as such are intended to 
be used by professional laboratory personnel trained in the 
competent handling of such materials. Responsibility for the safe 
use and disposal of products rests entirely with the purchaser and 
user. The Safety Data Sheet (SDS) for this product is available 
at www.waters.com.

VIII. ORDERING INFORMATION (Partial listing. For more information visit www.waters.com).

Description

Pore size

Particle size

Dimensions

Part no.

ACQUITY UPLC Glycoprotein BEH Amide

300Å

1.7 µm

2.1 x 5 mm Guard 3/pk with Std

176003699

ACQUITY UPLC Glycoprotein BEH Amide

300Å

1.7 µm

2.1 x 50 mm with Std

176003700

ACQUITY UPLC Glycoprotein BEH Amide

300Å

1.7 µm

2.1 x 100 mm with Std

176003701

ACQUITY UPLC Glycoprotein BEH Amide

300Å

1.7 µm

2.1 x 150 mm with Std

176003702

ACQUITY UPLC Glycoprotein BEH Amide

300Å

1.7 µm

2.1 x 100 MVK, 3/pk with Std

176003703

Glycoprotein Performance Test Standard

186008010

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ACQUITY UPLC Glycoprotein BEH Amide, 300Å, 1.7 μm Columns and Standard

Condition Your New Column

■

■

Column conditioning is a common practice used to optimize 
performance via passivation of secondary interactions.

■

■

HILIC of large molecules is a new frontier! Conditioning of new 
columns is as important as ever.

■

■

The amide bonding should be primed for its interaction with the 
analyte, yet once it is conditioned it need not be re-conditioned 
even after long-term storage.

TIP

■

■

Before using a new column, run 2 injections of your intended 
sample type at a mass load 10x greater than would be used in 
a normal analytical run.

■

■

Per conditioning run, load 20-100 μg protein or 
50-250 pmoles of released glycan.

■

■

If the column is for mAbs, use a mAb; tetrasialylated glycans, 
use a labeled/unlabeled fetuin N-glycan pool; etc.).

■

■

Run 1 blank and continue with your analyses.

Figure 6. Conditioning of a previously unused ACQUITY UPLC Glycoprotein BEH 
Amide, 300Å, 1.7 μm Column. Shown in the lower panel is an overlay of two 
analytical runs. 

EU 

0.00

5.00

10.00

15.00

20.00

25.00

30.00

35.00

40.00

45.00

50.00

6

EU 

0.00

5.00

10.00

15.00

20.00

25.00

30.00

35.00

40.00

45.00

50.00

8

10

12

14

16

18

20

22 min.

4

6

8

10

12

14

16

18

20

22 min.

4

After 1x

40 g Load 

Conditioning

After 2x

40 g Load 

Conditioning

HILIC FOR LARGE MOLECULES: P RACTICAL CONSIDERATIONS

Understand the Role of Ion Pairing

■

■

TFA ion pairing is often essential in large molecule HILIC 
separations not only because it improves the solubility of 
proteins in high ACN mobile phase but because it shields 
proteinaceous residues from interacting with the amide 
stationary phase.

■

■

TFA ion pairing therefore improves peak shape, selectivity 
and resolution of the hydrophilic residues and modifications 
of proteins and peptides.

■

■

The larger the proteinaceous analyte the more critical TFA 
ion pairing.

■

■

For smaller analytes, like peptides/glycopeptides, some TFA 
can be replaced with formic acid. However, peaks are likely to be 
more strongly retained and to exhibit additional broadening due 
to more complicated, heterogenous analyte to stationary phase 
interactions. Selectivity between aglycosylated and glycosylated 
peptides will also be affected. 

TIP

■

■

Use mobiles phases modified with 0.1% TFA whenever possible.

0.025

0.015

0.005

-0.005

8

9

10

11

12

13

14

15

16

17

18 min.

50 mM Ammonium Formate
pH 4.4

0.5% FA

0.1% TFA

TFA
Ion Pairing

Figure 7. Effect of mobile phase additive on the separation of ribonuclease B.

10

[ TIPS & TRICKS ]

ACQUITY UPLC Glycoprotein BEH Amide, 300Å, 1.7 μm Columns and Standard

Consider Your Diluents and Sample Solubility

■

■

It is common practice to match the composition of the 
sample diluent to your initial chromatographic conditions. 
HILIC of large molecules requires that this be given careful 
consideration, because some analytes may not be soluble 
in high organic concentrations. 70-90% ACN could be a 
condition that initiates the precipitation of your sample. 

■

■

Understand that HILIC can in fact be performed on aqueous 
samples. The only requirement is that very minimal volumes of 
sample be injected (see next section). 

■

■

High ACN diluents can be used, but care must be taken to 
enhance the solubility of the sample. 

TIP

■

■

Add TFA to the sample at concentrations between 0.2 to 1%.

■

■

Use additional additives, like heptafluorobutyric acid (HFBA) or 
hexafluoroisopropanol (HFIP).

■

■

Use a co-solvent, such as dimethylsulfoxide (DMSO), in 
concentrations ranging between 1 and 25%.

F

F

F

F

OH

O

F

F

F

O

S

H3C

H3C

H

H

H

C

C

N

F

O

F

F

OH

OH

F

F

F F F F

H

H

O

TFA
Ion Pairing

HFIP

DMSO

HFBA

How do you deal
with so much
organic solvent?

Can you load from
aqueous diluent?

If high ACN…

Figure 8. Additives to consider if using ACN/organic solvent diluents.

Minimize Injection Volumes

■

■

In most situations, HILIC injection volumes must be much 
lower than what can otherwise be used in reversed-phase 
chromatography.

■

■

100% AQUEOUS Samples

■

■

For a 2.1 mm ID column, maximum injection volumes are 
usually between 0.4 and 1.2 μL (depending on the dispersion 
of your LC).

■

■

ACETONITRILE Containing Samples

■

■

Adding ACN to a sample increases the allowable injection 
volume but can create solubility issues as noted earlier.

■

■

High ACN initial conditions can facilitate sample loading and the 
mixing of sample to higher ACN content before it sees the head 
of the column. Better band formation can thereby be achieved.

TIP

■

■

Empirically determine your maximum injection volume.

■

■

Inject varying volumes, i.e. 1, 3, and 10 μL.

■

■

Select an injection volume that does not show signs of 
breakthrough or solvent effects.

Figure 9. Determining the optimal volume load for an aqueous sample of ribonuclease 
B. An initial gradient condition to improve band formation (shown in red).

0.2

0.15

0.1

0.05

0

5

10

15

20 min.

Mass Load

1 µg

10 µL

2 µL

1 µL

0.5 µL

Peak distortion due
to solvent effects

Time (min)

%A

%B

0

33.0

67.0

20.0

40.0

60.0

21.0

100.0

0.0

23.0

100.0

0.0

24.0

33.0

67.0

34.0

33.0

67.0

Time (min)

%A

%B

0

15.0

85.0

0.5

15.0

85.0

1.0

33.0

67.0

21.0

40.0

60.0

22.0

100.0

0.0

24.0

100.0

0.0

25.0

15.0

85.0

35.0

15.0

85.0

11

[ TIPS & TRICKS ]

ACQUITY UPLC Glycoprotein BEH Amide, 300Å, 1.7 μm Columns and Standard

Figure 10. Using flow restriction post-column, pre-detector to eliminate the 
on-column aggregation of an intact mAb (trastuzumab).

Take Advantage of Temperature, Additives, and High 
Pressures to Avoid On-Column Aggregation 

■

■

With HILIC of large molecules, on-column aggregation can 
sometimes be encountered in the form of distorted, spurious 
peaks comprised of multiple analyte forms.

■

■

Choose method conditions that enhance the solubility of 
analytes in ACN.

■

■

Proteins are most soluble in ACN mixtures when in the 
presence of hydrophobic, strong acid mobile phase additives.

■

■

Elevated column temperatures improve solubility.

■

■

Ultrahigh back pressures can also be used to 
minimize aggregation.

TIP

■

■

Use hydrophobic mobile phase additives.

■

■

TFA and/or HFIP.

■

■

Avoid HFBA if performing LC-MS (adduct formation).

■

■

Explore elevated column temperatures if needed (i.e., 50 to 80 ˚C).

■

■

Use long column lengths, coupled columns and/or flow 
restrictors if possible.

0.2

0.1

0

20 min.

*Pressure at retention time of the mAb

15

10

7300 psi

Increasing
pressure

4500 psi

3200 psi

Aggregate peak

Flow restriction
25 µm I.D. PEEK tubing
post-column

Increasing column pressure
minimizes on-column aggregation

Disrupt Non-Covalent Interactions in Your Sample

■

■

The denaturing conditions of HILIC chromatography cannot 
always be relied upon to disrupt non-covalent interactions.

■

■

It is best practice to disrupt these interactions in the sample, 
prior to making an injection.

■

■

Non-covalent complexes can sometimes otherwise be separated 
to the detriment of accurately profiling a sample.

TIP

■

■

Add GuHCl to your sample (up to 6 M GuHCl into a fully 
aqueous sample).

Low Concentration 
Surfactant 

6M GuHCl 

No interference from 

non-covalent interactions 

0.45

0.40

0.35

0.30

0.25

0.20

A

214

0.15

0.10

0.05

0.00

15 min.

10

5

Heat denaturation

Self associating heavy chain subunits

Figure 11. HILIC profiling of reduced mAb samples prepared from various conditions. 

12

[ TIPS & TRICKS ]

ACQUITY UPLC Glycoprotein BEH Amide, 300Å, 1.7 μm Columns and Standard

Use a Flow Through Needle When Possible

■

■

With the possible solubility issues of proteins under HILIC 
conditions, it is best to use a flow through needle autosampler so 
that sample immediately goes to the LC flow path and is quickly 
pressurized via the pump and the column backpressure.

■

■

If restricted to a fixed loop system, use a full loop injection 
mechanism to eliminate issues with precipitation, which could 
occur as analyte is diluted at low pressure with needle wash 
during a partial loop injection.

■

■

Weak Needle Wash: 0.1% TFA, 85% ACN.

■

■

Strong Needle Wash : 0.1% TFA, water.

TIP

■

■

Use flow through needle injection mechanisms whenever possible.

Figure 12. Flow through needle injections.

Bottle tray

Detector

Column heater

Sample manager -
flow through needle

Solvent manager

Wash

Solvent

Purge

Solvent

Waste

Union

To

column

Inject valve

inject position

From solvent

manager

Flow Through

Needle

Wash pump

Solenoid

valve

Sample

To waste

Injection port

Sample compartment

IN

OUT

Pressure

transducer

Sample

syringe

13

[ TIPS & TRICKS ]

ACQUITY UPLC Glycoprotein BEH Amide, 300Å, 1.7 μm Columns and Standard

Waters, ACQUITY UPLC, UPLC, UltraPerformance LC, and The Science of What’s Possible are registered trademarks of Waters 
Corporation. eCord, VanGuard, GlycoWorks, and RapiFluor are trademarks of Waters Corporation. Acrodisc is a registered 
trademark of Pall Corporation. All other trademarks are the property of their respective owners. 

©2016 Waters Corporation. February 2016 Rev D  720005408EN  IH-PDF

Waters Corporation 
34 Maple Street 
Milford, MA 01757 U.S.A. 
T: 1 508 478 2000 
F: 1 508 872 1990 
www.waters.com

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