It depends on how your system is configured - primary considerations being
tubing length and tubing inner diameter (AKTA can come with at least three
different PEEK tube diameters, depending on what you purchased it for). New
systems often arrive with at least one tube-change kit (i.e. pre-cut tubes
of lengths suitable for direct replacement) - again, depending on the
options you purchased.
From the bottom of the column to the fraction collector
It's not at all hard to figure out the dead volume experimentally by setting
your column to a bypass and injecting a bolus of colored solution at the
same time as fraction collection begins (set your fractions to low volume
for added accuracy). As an added bonus by examining the absorption profile
of fractions on the edge of the peak you will also find out how much
dilution and mixing is introduced at the 'bottom end' of the system (it
should be rather small, if your system is correctly configured).
Notably, as Christian pointed out there should be a value set in Unicorn.
Depending on who did the installation and how long ago etc. that value may
or may not represent reality :)
The following method (from AKTA manual online) is a good, simple test
(accurate to around 100ul in my experience):
http://www.gelifesciences.com/aptrix/upp01399.nsf/Content/laboratory_support~laboratory_faq~faq~delay_volume?OpenDocumenthometitle=chromatography_support
*Method III - Determining the delay volume by balancing eluted water*
Manually set the flow path to the direction of the fraction collector.
Unscrew the tubing that is connected to inlet of the UV flow cell and insert
a luer adaptor instead. Fill a syringe with water and inject water into flow
cell unless it drops at the outlet of the fraction collector (in which case
you have likely exceeded the pressure in the tubing which might be more than
4 bar, depending on configuration and flow restrictor used). Now fill the
syringe with air (at least 20 ml because of compression) and displace the
water. Collect eluting water in a small cup. Determine the system delay
volume by balancing the cup before and after elution. Repeat two times for
calculation of a mean value. Enter the mean value in system settings in
UNICORN.
Artem
On Tue, Dec 21, 2010 at 9:17 AM, James Pauff pauf...@yahoo.com wrote:
Hello all,
Does anyone have a rough estimate (or has anyone actually determined) an
average dead/delay volume between buffers run on an AKTA Prime FPLC? We are
attempting to overexpress/isolate a smaller His-tagged transmembrane
protein, and require running several detergent buffers in succession over
the column. This obviously creates fractions that are just dead
volume/ddH2O between buffers, and we would like to narrow in on where to
look for the protein prior to analyzing the fractions (i.e. how many
fractions can we discard?). Should we run ddH2O and then the first mL into
'waste' before running each buffer over the column (and collecting
fractions)? Just not used to this system yet! Thank you!
Jim
