Waters Protein-Pak HR Ion-Exchange Glass Columns - Manual (Page 3)

[ Care and Use ManUal ]
Protein-Pak HR Ion-Exchanged Glass Columns 3
d. Choosing Sample Load Capacity
When choosing a column, consider the protein-binding capacity
of the packing material. Protein-binding capacity is generally 10
to 60 mg/mL of the packed bed for the Protein-Pak HR series of
packings. However, loading capacities vary for different samples.
For initial scouting conditions, load samples with a protein content
of less than 5% of total column capacity.
For preparative applications (described in Section I, e), protein
binding is affected by:
•
pH and ionic strength of the mobile phase
• Capacity of the ion exchanger
• Properties of sample molecules, for example, molecular weight,
isoelectric point, conformation, and other properties
• Competitive binding of compounds in the sample
c. Scaling
To scale up from analytical (Protein-Pak HR material in an AP Minicolumn
or AP-1) to preparative (Protein-Pak HR material in an AP-2 or AP-5) you
must adjust the following conditions:
•
Sample load
• Flow rate
• Gradient delay
To ensure predictable chromatography in the AP series of columns
during scale-up, use the same particle size and packing material as is
used in the preparative column.
Increasing sample load and flow rate
Use the following equation to increase sample load in proportion to
the volume of the packed bed:
L = column length
S = sample load
r = column radius
P = preparative
a = analytical
Use the following equation to increase flow rate in proportion to the
volume of the packed bed:
V = column volume
F = flow rate
L = column length
r = column radius
P = preparative
a = analytical
Similar retention times and peak widths for both the analytical and
the preparative column should be obtained, resulting in similar
resolution.
When the flow rate is scaled as recommended above, keep the
gradient time profile constant. However, it may be necessary to use
a lower flow rate because of pressure limitations on the preparative
instrument or the preparative column. In this case, adjust the gradi-
ent table as outlined below.
Adjusting gradient table/times
Adjust the gradient table when the flow rate is not scaled in propor-
tion to the column volume.
Use the following formula to:
Insert formula from scan
• Keep the ratio of the gradient volume to the column volume constant
• Adjust the gradient table
V = column volume
F = flow rate
t = times in gradient table
P = preparative
a = analytical
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