Number of Full Columns of Cartridges

This setting is critical to set the proper force used to mount the cartridges. To obtain expected instrument performance, ensure that the column selection is correct.

If the column selection is:

Default: 1

Range: 1-12

Initial Syringe Wash

This step flushes any potential contaminants from the syringes at the wash station before the cartridges are mounted.

During each Initial Syringe Wash cycle, the head aspirates 250 µL into the syringes from the wash station chimneys, and then moves by a fixed offset between the chimneys to dispense the syringe contents to waste.

This step is selected by default.

Wash Cycles. Increasing the number of wash cycles may clean the syringes better. However, more cycles increases the total run time and causes wear on the syringes.

Prime

This step removes entrained air from within the cartridges and properly wets the surface of the resin.

The Prime step aspirates the Priming Buffer into the syringes, mounts the cartridges, and then dispenses the buffer through the cartridges into the Organic Waste plate. The cartridges are parked at the seating station and the syringes are washed at the wash station.

The AssayMAP reversed-phase cartridges (C18, RPS, and RPW) used with the Peptide Cleanup application require a Priming Buffer containing at least 25% organic solvent. As illustrated in Agilent app note 5991-6478EN (Reference library), reversed-phase cartridges (C18, RPS, and RPW) that are primed with solutions containing a low percentage of organic solvent have greatly reduced binding capacity. Agilent recommends using a priming solution that contains at least 50% of an organic solvent such as acetonitrile.

This step is selected by default.

Volume (µL):

Flow Rate (µL/min):

Equilibrate

This step ensures that the resin bed is fully equilibrated with a solution that provides the optimal chemical conditions for binding during the Load Samples step.

During the Equilibrate step, the Equilibration Buffer is aspirated into the syringes, the cartridges are mounted, and then the buffer is dispensed through the cartridges into the wash station. The cartridges are parked at the seating station and the syringes are washed at the wash station.

The AssayMAP reversed-phase cartridges (C18, RPS, and RPW) typically used with the Peptide Cleanup application require an equilibration solution that has a very low concentration of or no organic solvent for effective binding during the sample loading step.

This step is selected by default.

Volume (µL). The default volume is equal to 10-column volumes, which should be sufficient for complete buffer exchange. Using less than the default volume may not fully equilibrate the resin bed. Using more than the default volume is unnecessary and increases run time.

Flow Rate (µL/min). A flow rate slower than the default will likely have no benefit, but will increase the total assay time. A flow rate faster than 20 µL/min using the default volume may not equilibrate through the pores in the beads.

Wash Cycles: The number of syringe wash cycles to perform at the end of this step. 250 µL of DI water is used for each syringe wash cycle.

Load Samples

This step allows the target analytes to bind to the surface chemistry of the resin bed.

This step mounts the cartridges on the syringes, aspirates the samples through the cartridges, and performs an external cartridge tip wash at the wash station to remove any sample on the outside of the cartridge tips. Then 5 µL of Equilibration Buffer (sample chase) is aspirated through the cartridges at the Load Samples flow rate. The sample chase ensures that the sample volume in the cartridges at the end of the sample load moves through the cartridge bed at the same rate as the rest of the sample. The cartridges are parked at the seating station and the flow-through plus sample chase are collected in the Flow Through Collection plate.

The protocol accommodates sample volumes up to 245 μL. Although, the form permits you to enter smaller volumes, the minimum advisable sample volume to be loaded onto an AssayMAP cartridge is 10 μL.

This step is selected by default.

Volume (µL). The volume of sample should be balanced with the sample concentration and the mass capacity of the cartridge. To determine the volume of sample to load, see Determining the volume of sample to load.

Flow rate (µL/min). The optimum sample loading flow rate requires balancing the speed of the assay and desired recovery. When setting the flow rate, be aware that the quantitative binding capacity is inversely proportional to the flow rate. Therefore, the maximum possible quantitative binding capacity is only obtained with very slow sample loading flow rates. If the amount of sample that you want to capture is significantly lower than the total possible qualitative binding capacity, you will be able to use a faster flow rate while maintaining quantitative binding.

Using flow rates slower than the default may not significantly increase analyte binding, and using flow rates faster than the default will decrease the quantitative binding capacity of the cartridges.

Wash Cycles. The number of syringe wash cycles to perform at the end of this step. 250 µL of DI water is used for each syringe wash cycle.

Cup Wash

This step removes the residual sample liquid that may remain above the resin bed after the Load Samples step.

The Cup Wash step aspirates Cartridge Wash Buffer into the syringes and then dispenses it into the cups of the parked cartridges. This liquid plus any residual liquid from the samples is aspirated from the cartridge cups. The protocol ensures that no cartridges are stuck to the probes before dispensing the liquid into the wash station at an offset from the chimneys, and then washing the syringes at the wash station.

This step is selected by default.

Volume (µL). Using a volume less than the default may be insufficient for cup washing, while using a volume >50 µL may offer little benefit.

Wash Cycles: Each wash cycle comprises one cup wash and one syringe wash.

Internal Cartridge Wash

This step uses Cartridge Wash Buffer to wash non-specifically bound molecules from the resin bed.

For the wash operation, this step mounts the cartridges, aspirates Cartridge Wash Buffer through the cartridges, washes the exterior of the cartridges, parks the cartridges, and then dispenses the contents of the syringes into the wash station at an offset from the chimneys. The syringes are then washed at the wash station.

Volume (µL). Volumes higher than the default volume (10 column volumes) may improve the purification marginally but also increases the run time. Volumes lower than the default volume may be insufficient for efficient cartridge washing.

Flow Rate (µL/min). A rate slower than the default flow rate will likely have little benefit, but will increase the total assay time. A rate faster than 20 µL/min may not equilibrate through the pores in the beads, resulting in incomplete washing.

Wash Cycles. The number of syringe wash cycles to perform at the end of this step. 250 µL of DI water is used for each syringe wash cycle.

Stringent Syringe Wash

This step cleans the syringes with the Syringe Wash Buffer prior to elution.

The Stringent Syringe Wash step aspirates the Syringe Wash Buffer and then dispenses the buffer into the Organic Waste plate. The syringes are then washed at the wash station.

This step is selected by default.

Volume (µL): Volumes higher than the default volume may improve the syringe cleaning but will increase the reagent consumption. Volumes lower than the default volume may be insufficient for efficient syringe washing.

Wash Cycles. A wash cycle is a stringent syringe wash followed by a basic syringe wash at the wash station.

Elute

This step uses Elution Buffer to elute bound peptides from the cartridges.

The Elute step aspirates the Elution Buffer into the syringes, washes the probes at the wash station, and then mounts the cartridges and dispenses the buffer through the cartridges at the specified flow rate into the Eluate Collection plate. An external cartridge tip wash is performed at the wash station to remove any sample on the outside of the cartridges, and then the cartridges are parked at the seating station.

After the elution, the samples are mixed in the Eluate Collection plate and the syringes are washed at the wash station.

This step is selected by default.

Volume (µL). The volume of Elution Buffer required for complete elution of bound analyte from the resin bed is dependent on the strength of the Elution Buffer. So the minimum elution volume must be determined empirically. The default volume is conservative and significantly higher than the minimum expected with a strong Elution Buffer.

Flow Rate (µL/min). A flow rate slower than the default is unlikely to improve the elution yield. Elution yield may be compromised if flow rates are faster than 15 µL/min for a given volume of elution buffer (that is, more buffer may be required to get the same elution yield at high flow rates relative to using lower flow rates for a given elution volume).

Wash Cycles. The number of syringe wash cycles to perform at the end of this step. 250 µL of DI water is used for each syringe wash cycle.

Re-Equilibrate

This step uses Equilibration Buffer to return the cartridge to a low percentage organic solvent condition.

The step aspirates the buffer into the syringes, mounts the cartridges, dispenses the buffer through the cartridges into the wash station, and then parks the cartridges.

Volume (µL). The default volume is equal to 10-column volumes, which should be sufficient for complete buffer exchange. Using less than the default volume may not fully equilibrate the resin bed. Using more than the default volume is unnecessary and increases run time.

Flow Rate (µL/min). A flow rate slower than the default rate will likely have no benefit, but will increase the total assay time. A flow rate faster than 20 µL/min using the default volume may not equilibrate through the pores in the beads.

Final Syringe Wash

This step uses the wash station to flush potential contaminants from the syringes.

During each Final Syringe Wash cycle, the head aspirates 250 µL of DI water into the syringes from the wash station chimneys, and then moves by a fixed offset between the chimneys to dispense the syringe contents to waste.

This step is selected by default.