Waters XBridge Protein BEH, C4, 300A, 3.5 µm Columns - Manual (Page 6)

6
[ CARE AND USE MANUAL ]
XBridge Protein BEH C
4
, 300Å, 3.5, 5, 10 �m
c. 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 can damage
the fluidic components of the HPLC system and will generally clog
the inlet distribution frit of the column. This will result in higher
operating pressure and poor performance.
d. Pressure
XBridge Protein BEH C
4
, 300Å, 3.5, 5, and 10 µm 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.
e. Temperature
Temperatures between 20–90 ˚C are recommended for operating
XBridge Protein BEH C
4
, 300Å 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.
IV. SCALING SEPARATIONS
Scale-Up Factors
Scale-up factor =
Consider scaling up from a 4.6 x 150 mm column to a
19 x 150 mm column:
Scale-up factor = = 17.1
Applying the scale-up factor, we can predict that approximately
17-135 mg of sample could be applied to the larger column (packed
with the same material as the analytical column). This range is
based on an analytical (4.6 mm I.D.) mass load of 1-8 mg.
Flow Rate
Flow rate (prep) = Flow rate (analytical) x
The calculated flow rate may be used for the larger column to ensure
the same linear velocity of mobile phases as used in the analytical
run. However, reasonable flow rates are based on column diameters.
Systems will be limited by increasing backpressure with increasing
column length and decreasing particle size.
Gradient Duration (GD)
GD (prep) =
Mass Loading
Approximate Mass Loading Capacity (mg) for Preparative OBD
Columns (Gradient Mode)
Diameter (mm)
Length (mm) 4.6 10 19 30
50 3 15 45 110
75 – – – 165
100 5 25 90 225
150 8 40 135 335
250 13 60 225 560
Reasonable Flow Rate (mL/min) 1.4 6.6 24 60
Reasonable Injection Volume (µL) 20 100 350 880
The calculated prep gradient duration is entered into the pump’s
gradient separation over the same number of column volumes as
was used in the analytical run.
Reasonable flow rates are based on column diameter. Systems will be
limited by increasing backpressure with increasing column length and
decreasing particle size.
Reasonable injection volumes are based on column diameter at a
length of 50 mm with relatively strong solvents. Increased length
is compatible with larger injection, but not proportionately so.
Weaker solvents significantly increase injection volume.
Mass loading capacities for peptide purifications depend strongly
on the sequence and may be estimated at 5–20% of listed values.
Many factors affect the mass capacity of preparative columns. The
listed capacities represent an ‘average’ estimate.
Capacity is:
§
Higher for strongly retained material
§ Higher for simple mixtures
§ Lower where higher resolution is required
(19) 2 x 150
(4.6) 2 x 150
(Diameter prep) 2 x Length prep
(Diameter analytical) 2 x Length analytical
GD (anal) x Length (prep)
x
Diameter (prep) 2
x
Flow Rate (analytical)
Length (analytical) Diameter (analytical) 2 Flow Rate (prep)
(Diameter prep) 2 Particle Size (analytical)
(Diameter analytical) 2 Particle Size (prep)
x