Waters Spherisorb Columns - Manual (Page 5)

[ Care and Use ManUal ]
Waters Spherisorb Columns 5
Table 3: Buffer Recommendations for Using Spherisorb Columns from pH 2 to 8
Additive or Buffer pK
a
Buffer
Range
(± pH unit)
Volatility
Used for
Mass Spec?
Comments
Formic Acid 3.75 Volatile Yes
Maximum buffering obtained when used with ammonium formate salt. Used in
0.1-1.0% range.
Acetic Acid 4.76 Volatile Yes
Maximum buffering obtained when used with ammonium acetate salt. Used in
0.1-1.0% range.
Ammonmium Formate
(NH
4
COOH)
3.75 2.75-4.75 Volatile Yes
Used in the 1-10 mM range for LC/MS. Higher concentrations (typically 20 mM)
are recommended for UV applications.
Note: sodium or potassium salts are not volatile.
Trifluoroacetic Acid
(TFA)
0.3 Volatile Low Conc.
When used in LC/MS, due at signal suppression, it is generally recommended to
use TFA at coencentrations <0.1%.
Ammonium Acetate
(CH
3
COONH
4
)
4.76 3.76-5.76 Volatile Yes
Used in the 1-10 mM range for LC/MS. Higher concentrations (typically 20 mM)
are recommended for UV applications.
Note: 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 guard column to maxmize
lifetime.
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.
(Please refer to the filtration section of the Waters Chromatography
Columns and Supplies Catalog or the Waters web site (www.waters.com)
for additional product information.) 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 poorer 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
Spherisorb columns can tolerate pressures of up to 6,000 psi (400 bar
or 40 MPa) although long-term, routine operating pressures greater
than 4,000 – 5,000 psi should be avoided in order to maximize column
and system lifetimes.
f. Temperature
Temperatures between 20 – 45 ˚C are recommended for operating
Spherisorb columns in order to enhance selectivity, lower
solvent viscosity and increase mass transfer rates. However, any
temperature above ambient may have a negative effect on lifetime
which will vary depending on the pH and buffer conditions used.
iii. scalinG up/doWn isocratic methods
The following formulas will allow scale up or scale down, while
maintaining the same linear velocity, and provide new sample
loading values:
If column i.d. and length are altered:
F
2
= F
1
(r
2
/r
1
) 2
or
Injection volume
1
= Injection volume
2
(r
2
/r
1
) 2 (L
2
/L
1
)
Where: r = radius of the column, in mm
F = flow rate, in mL/min
L = length of column, in mm
1 = original, or reference column
2 = new column