Waters XSelect CSH HPLC Columns - Manual (Page 4)

[ CARE AND USE MANUAL ]
4 XSelect CSH HPLC Columns
Table 3: Buffer Recommendations for Using XSelect CSH columns up to pH 11
d. Solvents
To maintain maximum column performance, use high quality chromatography grade solvents. Filter all aqueous buffers prior to use. Pall
Gelman Laboratory Acrodisc
® filters are recommended. Solvents containing suspended particulate materials will generally clog the outside
surface of the inlet distribution frit of the column. This will result in higher operating pressure and poor performance.
Degas all solvents thoroughly before use to prevent bubble formation in the pump and detector. The use of an on-line degassing unit is also
recommended. This is especially important when running low pressure gradients since bubble formation can occur as a result of aqueous
and organic solvent mixing during the gradient.
e. Pressure
XSelect CSH columns can tolerate pressures of up to 6,000 psi (400 bar or 40 Mpa) although pressures greater than 4,000 – 5,000 psi
should be avoided in order to maximize column and system lifetimes.
f. Temperature
Temperatures up to 80 ˚C are recommended for operating XSelect CSH columns in order to enhance selectivity, lower solvent viscosity and
increase mass transfer rates. However, any temperature above ambient will have a negative effect on lifetime which will vary depending on
the pH and buffer conditions used. See Table 2 for recommended pH and temperature operating ranges.
Additive/Buffer pKa Buffer range Volatility
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.
Phosphate 3 12.3 11.3 - 13.3 Non-volatile No Above pH 7, reduce temperature/concentration and use a guard column to maximize lifetime.
4-Methylmorpholine ~8.4 7.4 – 9.4 Volatile Yes Generally used at 10 mM or less.
Ammonia (NH
4
OH)
Ammonium Bicarbonate
9.2
10.3 (HCO
3
- )
9.2 (NH
4
+ )
8.2 – 10.2
8.2 – 11.3 Volatile
Volatile Yes
Yes
Used in the 5-10 mM range (for MS work keep source >150 ˚C ). Adjust pH with ammonium
hydroxide or acetic acid. Good buffering capacity at pH 10.
Note: use ammonium bicarbonate (NH
4
HCO
3
), not ammonium carbonate ((NH
4
)
2
CO
3
)
Ammonium (Acetate) 9.2 8.2 – 10.2 Volatile Yes Used in the 1-10 mM range.
Ammonium (Formate) 9.2 8.2 – 10.2 Volatile Yes Used in the 1-10 mM range.
Borate 9.2 8.2 – 10.2 Non-volatile No Reduce temperature/concentration and use a guard column to maximize lifetime.
CAPSO 9.7 8.7 – 10.7 Non-volatile No Zwitterionic buffer, compatible with acetonitrile, used in the 1-10 mM range. Low odor.
Glycine 2.4, 9.8 8.8 – 10.8 Non-volatile No Zwitterionic buffer, can give longer lifetimes than borate buffer.
1-Methylpiperidine 10.2 9.3 – 11.3 Volatile Yes Used in the 1-10 mM range.
CAPS 10.4 9.5 – 11.5 Non-volatile No Zwitterionic buffer, compatible with acetonitrile, used in the 1-10 mM range. Low odor.
Triethylamine
(as acetate salt)
10.7 9.7 – 11.7 Volatile Yes
Used in the 0.1-1.0% range. Volatile only when titrated with acetic acid (not hydrochloric or
phosphoric). Used as ion-pair for DNA analysis at pH 7-9.
Pyrrolidine 11.3 10.3 – 12.3 Volatile Yes Mild buffer, gives long lifetime.