Waters Sep-Pak Cartridges and Plates - Manual (Page 4)

[ Care and Use ManUal ]
Sep-Pak Cartridges and Plates 4
5. Elution Step
Once the interferences are washed off the cartridge, a strong
solvent is introduced to elute the analytes of interest. The volume
and flow rate of the eluting solvents should be precisely controlled as
in the load step to ensure reproducible results.
Table 2. Guidelines on the various types of separation mechanisms
V. storAGe And dIsPosAL oF used CArtrIdGes
Cartridges stored in their original sealed pouch remain stable for long
periods. To store unused cartridges in opened pouches, squeeze the air out
of the pouch, fold over the open end of the pouch twice, seal with tape, and
store in a desiccator.
Dispose of used cartridges safely in accordance with applicable government
or local regulations.
3. Load Step
When the analytes of interest are not retained by the sorbent, this is
called analyte breakthrough. For some methods, such as pass-through
cleanup, analyte breakthrough is desirable and is maximized for
those specific methods. However, in all other cases, analyte break-
through is unwanted and contributes to poor recovery and method
reproducibility. Breakthrough occurs when:
• There is too high an organic concentration in the load
solution for very polar analytes. Dilute sample at least 1:1
with water or buffer prior to loading.
• The analytes are bound to proteins, they may pass through
the sorbent. Ensure that analytes are not bound to proteins
by acidifying or basifying the sample.
• Sorbent is overloaded by the matrix component.
Therefore, it is important to choose the correct sorbent
mass (see Table 1).
• The flow rate of the load step is too fast. There is not
enough contact time between the analytes and the sorbent.
Look at the drops and adjust the vacuum so that you see
discrete droplets, not a stream of liquid.
Table 1. Choice of cartridges based on sample size
* LP=large particules (60 µm)
4. Wash Step
The wash steps are designed to remove unwanted matrix components
that remain from the loading step. The ideal wash solvent removes
only the matrix while keeping the analytes bound to the sorbent. For
complex samples this is impossible, so the wash steps are optimized
using pH, solvent strength, and solvent polarity to remove as much
matrix as possible while maintaining acceptable analyte recovery.
Sample Size Cartridge
1 - 10 mL 1 cc/30 mg or 3 cc/60 mg
10 - 100 mL 3 cc/60 mg or 6 cc/200 mg
100 - 500 mL 6 cc/200 mg or 6 cc/500 mg (LP*)
500 - 1000 mL 6 cc/500 mg (LP) or 12 cc/1 g (LP*)
Reversed Phase Normal Phase Ion Exchange
Analyte Low to
moderate
polarity/
hydrophobic
Moderate to
highly polar/
uncharged
Charged
or ionized
Matrix Aqueous Non-polar
organic solvent
Aqueous/low
ionic strength
Condition/
Equilibrate 1. Solvate polar
organic
2. Water
Non-polar
organic
Low ionic
strength buffer
Wash Aqueous/buffer Non-polar Low ionic
strength buffer
Elution
Steps Increase polar
organic content
in steps
Increase
moderate to
high polarity
organic content
in steps
Stronger
buffers–ionic
strength or pH
to neutralize
the charge