Waters Oligonucleotide Separation Technology Standard User Manual

[ Care and Use ManUal ]

massPreP olIgonuCleotIde seParatIon teChnology standard

I. IntroduCtIon

The pre-packaged MassPREP™ Oligonucleotide Separation
Technology (OST) Standard is designed for verification of HPLC/

®

instrument and column performance for analysis of synthetic

UPLC
oligonucleotides. Approximately equimolar amounts of 15, 20, 25,
30 and 35 nucleotide (nt) long oligodeoxythymidines are lyophilized
and packaged in 1.5 ml LC vials. These vials are vacuum-sealed in
foil pouches to reduce degradation that can occur by excessive expo­sure to light and air. Approximately 1 nmole of each oligonucleotide
is present in the vial.

II. reCommended usage

Waters MassPREP OST standard is intended for testing the column and
HPLC/UPLC systems separation performance. For selection of Waters

®

XBridge™ OST C18, 2.5 µm HPLC or ACQUITY UPLC
UPLC column, see document 720002008EN on waters.com/library. Both
columns and LC system performance can be tested using this dedicated
MassPREP OST standard. The most common problems in oligonucleotide
analysis that can be detected using OST standard are listed in the Table 1.

OST C18, 1.7 um

Contents

I. IntroduCtIon

II. reCommended usage

III. PreParatIon ProCedure

IV. storage and stabIlIty

V. orderIng InformatIon

MassPREP Oligonucleotide Separation Technology Standard 1

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Table 1: System Troubleshooting using MassPREP OST
Standard

Chromatogram Appearance Potential problem

Peaks elute outside of
the expected retention
time window

Tailing peaks Poor tubing connections (especially

Inconsistent peak width,
split peaks

Lost resolution A ge d or c on t am i na t e d c ol u m n.

Incorrectly prepared mobile phase,
aged mobile phase, incorrect col­umn temperature setting; excessive
gradient delay (method transfer
between different LC systems).

between column and detector);
column bed deterioration, column
contamination.

Inadequate gradient mixing,
incompatible sample solvent or
weak wash (purge solvent for

®

Alliance

(Has column backpressure
changed?)

HT).

III. PreParatIon ProC edure

The following procedure is provided as a general guideline for
MassPREP OST standard reconstitution. The described method
only serves as a starting point. Depending on the specific
application, one may consider using other solvents and/or dilutions.
Recommended chemicals to prepare sample diluent and LC mobile
phase are following: Acetic Acid (2M):Triethylamine (2M), Fluka,
P/N 09748; 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), Fluka, P/N
52512; triethylamine (TEA), Sigma, P/N 417283.

3. For MassPREP OST standard analysis in UPLC mode, prepare the
following mobile phases:

a. Mobile phase A is 15 mM TEA, 400 mM HFIP in water. Add

8.31 mL (13.44 g) of HFIP into 191.3 g of water, add 416 µL of
TEA; the final volume is 200 mL, the buffer pH is ~7.9.

b. Mobile phase B is 50% A, 50% MeOH. Prepare TEA-HFIP buffer

as described above, and add 158.2 g of MeOH (200 mL).

c. T he LC solvents are highly volatile; the mobile phase should

be used only 24 hours only before making a fresh one. Keep
the mobile phase containers sealed to minimize the buffer
evaporation.

4. Prime the ACQUITY UPLC system and connect a 2.1 x 50 mm
ACQUITY UPLC OST C18, 1.7 µm column (P/N 186003949). Setup
flow rate at 0.2 mL /min, and column temperature at 60 °C. Equilibrate
with initial mobile phase conditions (38% B) for ~20 minutes.

5. Inject 10 µL of the MassPREP OST standard. Run the gradient from 38
to 50% B in 12 minutes. The example of chromatogram is shown in
Figure 1.

6. The MassPREP OST sample can also be used to troubleshoot XBridge
OST C18, 2.5 µm columns configured to a HPLC system. The resolution
is not expected to match the performance obtained with a ACQUIT Y
UPLC OST C18, 1.7 µm column and UPLC System.

7. The alternative conditions for HPLC (UPLC) oligonucleotide analysis
use TEAA ion-pairing system:

a. Mobile phase A is 100 mM TEAA (measure 190 g of water,

add 10 mL of 2M:2M Acetic Acid:Triethylamine Stock Solution.
The pH adjustment is not necessary.

b. Mobile phase B: 20% acetonitrile, 80% TEAA. Prepare 200 mL

of TEAA as above, and add 50 mL (39.3 g) of acetonitrile.

1. Prepare 100 mM of triethylammonium acetate (TEAA) by diluting a
2M:2M stock solution of Acetic Acid:Triethylamine 20 fold in deionized
or HPLC grade water.

2. Add 0.5 mL of 100 mM TEAA in MassPREP OST standard vial. T he
final concentration is ~2 pmole/µL for each oligonucleotide. Vortex the
vial briefly to dissolve and homogenize the sample.

MassPREP Oligonucleotide Separation Technology Standard 2

c. T he most useful analytical column for HPLC is a 2.1 x 50 mm,

XBridge OST C18, 2.5 µm column (P/N 186003952). Flow rate
is set to 0.2 mL/min, column temperature to 60 °C. Gradient
is 40-60% B in 25 minutes. Approximately 20-40 µL of the
MassPREP OST sample is typically injected on column (80 pmole
per peak). Example of chromatogram is not shown.

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0 mi nutes 10

15 nt

20 nt

25 nt

30 nt

35 nt

V. orderIng InformatIon

Figure 1: ACQUITY UPLC analysis of MassPREP OST standard on an
ACQUITY UPLC OST C18, 1.7 µm column. The main components are
labeled. Small peaks eluting between labeled oligonucleotides are N-1,
N-2, etc. failure sequences generated during the oligonucleotide syn­theses. The ACQUITY UPLC system is equipped with 50 µL standard

mixer and PDA detector (260 nm).

IV. storage and stabIlIty

It is recommended that the MassPREP OST Standard remain in
the foil pouch (in the dark) at room temperature until use. Once
reconstituted in appropriate solvents, the MassPREP OST Standard
solution is stable for one week when stored at 4 ˚C. Stability can be
extended by freezing samples at -20 ˚C.

Description

ACQUITY UPLC
OST C18 *

ACQUITY UPLC
OST C18 *

Custom
ACQUITY UPLC
OST C18 *
XBridge OST
C18
XBridge OST
C18
XBridge OST
C18

Custom XBridge
OST C18
MassPREP OST

Standard

* For use on Waters ACQUITY UPLC Systems

† MSDS information: Waters MassPREP OST Standard contains a non
hazardous peptide in powder form. It is for laboratory use only; solely
for purposes of scientic experimentation, analysis, research or develop­ment. It should be used only as directed and in accordance with good
laboratory practice. It is non hazardous as dened by OSHA’s Hazard
Communication Standard, 29 CFR 1910.1200(c) and Appendix A and B,
and are thus exempt from the requirements of the Hazard Communica­tion Standard, including the requirement to supply MSDSs.

Particle

Size

1.7 μm 135 Å 2.1 x 50 mm 186 00 394 9

1.7 μm 135 Å 2.1 x 100 mm 18 60 039 50

-- -- -- 186 00 3951

2.5 μm 135 Å 2.1 x 50 mm 186 003 952

2.5 μm 135 Å 4.6 x 50 mm 18 600 395 3

2.5 μm 135 Å 10 x 50 mm 186003954

-- -- -- 186 003 955

Pore
Size

Dimension Part No.

1 Vial 186004135

MassPREP Oligonucleotide Separation Technology Standard 3

[ Care and Use ManUal ]

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© 2007 Waters Corporation. Waters, The Science of W hat’s
Possible, MassPREP, XBridge, ACQUITY, Alliance and UPLC are
trademarks of Waters Corporation.

October 2007 715001677 Rev A VW-P DF

MassPREP Oligonucleotide Separation Technology Standard 4

Waters Corporation

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