Waters Oligonucleotide Separation Technology ACQUITY UPLC C18 Columns - Manual (Page 4)

[ Care and Use ManUal ]
4
2) Add 5.6 mL of glacial Acetic Acid into 950 mL of water and
mix well.
3) Slowly add 13.86 mL of TEA.
4) The pH should be adjusted to pH 7 +/- 0.5 by careful addition
of Acetic Acid.
5) Adjust final volume to 1 L with water.
Alternatively, premixed TEAA can be used [e.g., Sigma 1 M TEAA
(part # 90357)]. Mix 100 mL with 900 mL of water to prepare 1 L of
0.1 M TEAA mobile phase.
Alternative ion-pairing reagents are recommended for improved sepa-
ration of phosphorothioates or when performing LC-MS analyses. An
ion-pairing mobile phase based on Triethylamine (TEA) and Hexafluo-
roisopropanol (HFIP) as the buffering acid produces an efficient eluent
system for improved separations involving these application types.
As indicated below, two ion-pairing systems are useful.
For routine detritylated oligonucleotide applications, aqueous buffer
consisting of 8.6 mM TEA and 100 mM HFIP is effective. For applica-
tions such as those involving the separation of G-rich oligonucle-
otides, it is advisable to use aqueous buffer consisting of 15 mM TEA
and 400 mM HFIP (pH 7.9).
tea-hfIp System 1
1L of 8.6 mM TEA / 100 mM HFIP is prepared as follows:
1) Perform work in a hood
2) Add 10.4 mL of HFIP (16.8 g) into 988.4 g of water
and mix well.
3) Slowly add 1.2 mL of TEA.
4) The pH is approximately 8.3 +/- 0.1.
tea-hfIp System 2
1 L of 15 mM TEA / 400 mM HFIP is prepared as follows:
1) Perform work in a hood
2) Add 41.56 mL (67.17 g) of HFIP into 956.36 g of water
and mix well.
3) Slowly add 2.08 mL (1.52 g) of TEA.
4) The pH of final buffer is approximately 7.9 +/- 0.1.
c. recommended Injector Wash Solvents
Between analyses, the ACQUITY UPLC
® system injector and seals
can and should be washed with two separate solvents. A 90%
water/10% acetonitrile mixture is the recommended strong solvent
injector wash solution for the TEAA ion-pairing based method.
A 90% water/10% methanol mixture is the recommended strong
solvent injector wash solution for the TEA-HFIP based method.
0.20 μm membrane filtered, LC grade Water is the recommended
weak wash solvent solution for all ACQUITY OST separation methods.
Note: Do not use OST Separation mobile phases A and B for the
respective weak and strong injector wash solvents especially with TEA-
HFIP ion pairing systems due to seal incompatibility issues with HFIP.
d. ph range
The recommended operating pH range for ACQUITY UPLC
® OST C
18
columns is 1 to 12.
e. pressure
ACQUITY UPLC
® OST C
18
columns can tolerate pressures of up to
15,000 psi (1034 bar or 103 Mpa).
f. temperature
Temperatures between 20 ˚C – 65 ˚C are recommended for operating
ACQUITY UPLC
® OST C
18
columns in order to enhance selectivity,
lower solvent viscosity and increase mass transfer rates.
Note: Operating at elevated pH, temperature, and/or pressure may
potentially result in shortened column life.
g. aCQuIty uplC ® mixer options
The standard Waters ACQUITY UPLC ® system is equipped with 50 µL
in-line mobile phase mixer. For demanding biopolymer separations
(e.g., peptide mapping), use of a shallow gradients (e.g., 0.15%
Mobile Phase B change per min) is required. In these situations, it is
recommended that the organic solvent concentration in Mobile Phase
B be reduced by “premixing” with a measured amount of mobile
phase A (e.g., mobile phase A= 0.1 M TEAA and mobile phase B=
Acetonitrile / 0.1M TEAA, 20/80, v/v).
Use of a 425 µL mixer (Part # 205000403) specifically designed
for shallow UPLC
® gradient separations is recommended when the
solvent premixing technique (detailed above) is not used and when
mobile phase B contains either 100% acetonitrile (for TEAA ion-pair-
ing method) or 100% methanol (for TEA-HFIP ion-pairing method).
In addition, the Solvent Deliver System Outlet Tube Assembly (Part