Method guidelines – Waters Oligonucleotide Separation Technology ACQUITY UPLC C18 Column User Manual

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[ method guidelines ]

1: Standard oligonucleotides: 10 – 30 mers
2: Short oligonucleotides: Less than 10 mer
3: Long oligonucleotides: 30 – 60 mers
4: Assuming use of a 2.1 x 50mm ACQUITY UPLC

OST C

column

at a flow of 0.2 mL/min and a separation temperature of 60 ˚C.

The retention of dye labeled and dually-labeled oligonucleotides is
dictated by the nature of label. For example, the retention of 25 mer
oligonucleotide increases according to the type of label attached as
follows: no label<6FAM<

VII. AnALYsIs oF ModIFIed oLIgonUCeUtIdes

ACQUITY UPLC

OST C

columns are suitable for analysis of unmodi-

fied as well as modified detritylated oligodeoxyribonucleotides and
oligoribonucleotides. Phosphorothioate and/or 2’-O -alkyl modified
oligonucleotides can also be analyzed with IP-RP-HPLC method. The
modified oligonucleotides are usually more difficult to resolve from
N -1 etc. impurities. The recommended ion-pair system for phospho-
rothioate oligonucleotide analysis is TEA-HFIP (see Recommended
mobile phases).

Peptide nucleic acids (PNA) can also be analyzed using ACQUITY
UPLC

OST C

columns. The ion-pairing system recommended for

analysis of PNA is similar to those used for peptide analysis (0.1%
Trifluoroacetic acid or formic acid).

Gradient 1

[standard oligonucleotides (1)]

Gradient 2

[High GC content or short oligonucleotides (2)]

Gradient 3

[High AT content or long oligonucleotides (3)]

Initial % ACN

7.00%

5.25%

7.50%

Final % ACN

10.75%

9.00%

12.50%

Gradient Length(4)

15 min

20 min

Table 2: Suggested Gradient Conditions for Non-standard Detritylated Synthetic Oligonucleotide Sequences

Waters Part Number

Column Name

Pore Size

Particle Size

Dimension

186003949

*ACQUITY UPLC

OST C

135Å

1.7 micron

2.1 x 50 mm

186003950

*ACQUITY UPLC

OST C

135Å

1.7 micron

2.1 x 100 mm

186003951

*Custom ACQUITY UPLC

OST C

Table 3: Ordering Information

*Note: Optimum performance of ACQUITY UPLC

OST C

columns is best assured using an appropriately configured Waters ACQUITY UPLC

System.

Consequently, use of ACQUITY UPLC

OST C

columns on conventional HPLC systems is not recommended.