Waters ACQUITY UPLC HSS Columns User Manual | Page 4 / 9

[ Care and Use ManUal ]

XSelect HSS HPlc columnS

4

and 1 to 8 for the HSS c

18

chemistry. A listing of commonly used

buffers and additives is given in table 2. Additionally, the column lifetime will vary depending upon the operating temperature, the type and concentration of buffer used.

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

c. Solvents

to maintain maximum column performance, use high quality chro-matography grade solvents. Filter all aqueous buffers prior to use through a 0.2 µm filter. Solvents containing suspended particulate materials will generally clog the outside surface of the inlet distri-bution frit of the column. this will result in higher operating pres-sure and poorer performance. See Section V for more information.

d. Pressure

AcQuItY uPlc HSS columns can tolerate operating pressures up to 18000 psi (1241 bar or 124 mPa).

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

e. Temperature

temperatures between 20–45 °c are recommended for operating AcQuItY uPlc HSS columns in order to enhance selectivity, lower solvent viscosity and increase mass transfer rates. When operating at high pH, lower operating temperatures are recommended for longer column lifetime. Working at high temperatures (e.g. > 40 ˚c) may also result in shorter column lifetimes.

Table 2: Buffer Recommendations for Using ACQUITY UPLC HSS Columns

Additive/Buffer

pKa

Buffer

Range

Volatility

(±1 pH unit)

Used for

Mass Spec

Comments

TFA

0.3

Volatile

Yes

Ion pair additive, can suppress mS signal, used in the 0.02-0.1% range.

Acetic Acid

4.76

Volatile

Yes

maximum buffering obtained when used with ammonium acetate salt. used in 0.1-1.0% range.

Formic Acid

3.75

Volatile

Yes

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

Acetate (NH

4

CH

2

COOH)

4.76

3.76 – 5.76

Volatile

Yes

used in the 1-10 mm range. note that sodium or potassium salts are not volatile.

Formate (NH

4

COOH)

3.75

2.75 – 4.75

Volatile

Yes

used in the 1-10 mm range. note that sodium or potassium salts are not volatile.

Phosphate 1

2.15

1.15 – 3.15 non-volatile

no

traditional low pH buffer, good uV transparency.

Phosphate 2

7.2

6.20 – 8.20 non-volatile

no

Above pH 7, reduce temperature/concentration and use a guard column to maximize lifetime.

II. Column use

to ensure the continued high performance of AcQuItY uPlc HSS columns, follow these guidelines:

a. Sample Preparation

4. Sample impurities often contribute to column contamination.

one option to avoid this is to use Waters oasis

®

solid-phase

extraction cartridges/columns or Sep-Pak

®

cartridges of the

appropriate chemistry to cleanup the sample before analysis. For more information, visit www.waters.com/sampleprep

5. It is preferable to prepare the sample in the operating mobile

phase or a mobile phase that is weaker than the mobile phase for the best peak shape and sensitivity.

6. If the sample is not dissolved in the mobile phase, ensure that

the sample, solvent and mobile phases are miscible in order to avoid sample and/or buffer precipitation.

Filter sample with 0.2 µm membranes to remove particulates. If the sample is dissolved in a solvent that contains an organic modi-fier (e.g., acetonitrile, methanol, etc.) ensure that the membrane material does not dissolve in the solvent. contact the membrane manufacturer with solvent compatibility questions. Alternatively, centrifugation for 20 minutes at 8000 rpm, followed by the transfer of the supernatant liquid to an appropriate vial, could be considered.

b. pH Range

the recommended operating pH range for AcQuItY uPlc HSS columns is 2 to 8 for the t3, c

18

SB, cyano and PFP chemistries,