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ECL`

ExperimentLiquidLiquidExtraction

ExperimentLiquidLiquidExtraction[Samples]⟹Protocol

creates a Protocol object to separate the aqueous and organic phases of Samples via pipette or phase separator, in order to isolate a target analyte that is more concentrated in either the aqueous or organic phase.

    
Liquid Liquid Extraction (LLE) separates the aqueous and organic phases of given samples via pipette or phase separator, in order to isolate a target analyte that is more concentrated in either the aqueous or organic phase.
    

Experimental Principles

    Figure 1.1: Procedural overview of a Liquid Liquid Extraction experiment that uses a Phase Separator to separate the Aqueous/Organic layers. Step 1: The input sample, Demulsifier, and the OrganicSolvent are mixed and the biphasic solution is transferred into the Phase Separator cartridge. Step 2: The organic phase of the biphasic solution (which is denser than the aqueous phase) passes through the hydrophobic frit at the bottom of the phase separator cartridge, draining into a collection container below the cartridge.
    Figure 1.2: Procedural overview of a Liquid Liquid Extraction experiment that uses a Pipette to separate the Aqueous/Organic layers. Step 1: Organic and/or Aqueous solvent is added to the ExtractionContainer to create a biphasic solution (optionally, with the aid of Demulsifier). Step 2: Mixing via pipette or shaking is performed to promote the separation of the TargetAnalyte of interest into the TargetPhase. Step 3: After separation, Aqueous and Organic layers are aliquotted into their specified ImpurityContainerOut / TargetContainerOut. The position of the boundary layer is estimated from the volumes of AqueousSolvent / OrganicSolvent that is added to the sample. To control the position of the boundary layer, use the ExtractionTransferLayer and ExtractionObjective options.

Instrumentation

    STARlet

    Figure 2.1: Overhead view of the liquid handling instrument deck.

    Super STAR

    Figure 2.2.1: An overview of the robotic workcell. It is capable of processing plate operations including incubation, shaking, and magnetic bead separation on-deck and centrifugation, filtration, and absorbance/fluorescence/luminescence measurement off-deck, with a robot arm facilitating the plate movements.
    Figure 2.2.2: Overhead view of the liquid handling instrument deck.

Experiment Options

    General

    ExtractionTechnique

    The method that is used to separate the aqueous and organic phase of a sample. The collection of the target phase occurs after the extraction solvent(s) and demulsifier (if specified) are added, the sample is mixed (optionally), allowed to settle for SettlingTime (for the organic and aqueous phases to separate), and centrifuged (optionally). Pipette uses a pipette to aspirate off either the aqueous or organic layer, optionally taking the boundary layer with it according to the IncludeBoundary and ExtractionBoundaryVolume options. PhaseSeparator uses a column with a hydrophobic frit, which allows the organic phase to pass freely through the frit, but physically blocks the aqueous phase from passing through. Note that when using a phase separator, the organic phase must be heavier than the aqueous phase in order for it to pass through the hydrophobic frit, otherwise, the separator will not occur.
    Default Value: Automatic
    Default Calculation: If the option ExtractionDevice is set, ExtractionTechnique is set to PhaseSeparator (this option only applies to the use of a phase separator). If the options IncludeBoundary or ExtractionBoundaryVolume are set, ExtractionTechnique is set to Pipette (these options only apply to the use of a pipette for separation). Otherwise, set to PhaseSeparator by default.
    Pattern Description: Pipette or PhaseSeparator.
    Programmatic Pattern: ExtractionTechniqueP | Automatic
    Index Matches to: experiment samples

    ExtractionDevice

    The device which is used to physically separate the aqueous and organic phases.
    Default Value: Automatic
    Default Calculation: If ExtractionTechnique is set to PhaseSeparator, ExtractionDevice will be set to Model[Container, Plate, PhaseSeparator, "Semi-Transparent Plastic 96 Fixed Well Plate with Phase Separator Frits"].
    Pattern Description: An object of type or subtype Model[Container, Plate, PhaseSeparator] or Object[Container, Plate, PhaseSeparator] or a prepared sample or Null.
    Programmatic Pattern: ((ObjectP[{Model[Container, Plate, PhaseSeparator], Object[Container, Plate, PhaseSeparator]}] | _String) | Automatic) | Null
    Index Matches to: experiment samples

    SelectionStrategy

    Indicates if additional rounds of extraction are performed on the impurity phase (Positive) or the TargetPhase (Negative). Positive selection is used when the goal is to extract the maximum amount of TargetAnalyte from the impurity phase (maximizing yield). Negative selection is used when the goal is to remove impurities that may still exist in the TargetPhase (maximizing purity).
    Default Value: Automatic
    Default Calculation: If NumberOfExtractions is set to 1, then set to Null (this option only applies if there are multiple rounds of extraction). If SolventAdditions or AqueousSolvent/OrganicSolvent is specified, then the solvent(s) specified are used to infer the SelectionStrategy (if the phase of the solvent being added matches TargetPhase, then SelectionStrategy is set to Positive (extraction is done on the impurity layer), otherwise SelectionStrategy is set to Negative). Otherwise, automatically set to Positive selection.
    Pattern Description: Positive or Negative or Null.
    Programmatic Pattern: (SelectionStrategyP | Automatic) | Null
    Index Matches to: experiment samples

    IncludeBoundary

    Indicates if the boundary layer is aspirated along with the ExtractionTransferLayer. This option is only applicable when ExtractionTechnique is set to Pipette.
    Figure 3.1: The ExtractionTransferLayer indicates the layer (Top or Bottom) that should be removed from the biphasic solution if ExtractionTechnique->Pipette (does not apply to phase separators). The boundary between the layers will be aspirate along with the ExtractionTransferLayer if IncludeBoundary -> True. Note that if ExtractionTransferLayer matches TargetPhaseLayer (the TargetPhase is being aspirated), setting IncludeBoundary->True will maximize yield, otherwise, setting IncludeBoundary->False will maximize purity. The inverse is true if ExtractionTransferLayer does not match TargetPhaseLayer (the Impurity phase is being aspirated).
    Default Value: Automatic
    Default Calculation: Automatically set to False if ExtractionTechnique is set to Pipette. Otherwise, set to Null since IncludeBoundary does not apply when ExtractionTechnique is PhaseSeparator since the hydrophobic frit of the phase separator automatically will only allow the organic phase to pass through.
    Pattern Description: List of one or more True or False entries or Null.
    Programmatic Pattern: ({BooleanP..} | Automatic) | Null
    Index Matches to: experiment samples

    TargetAnalyte

    The desired molecular entity that the extraction is designed to isolate.
    Default Value: Automatic
    Default Calculation: Automatically set to the value of Analytes field in the input sample, if the Analytes field is populated. Otherwise, set to the first element of the Composition field that's listed in terms of concentration (molarity) or mass concentration (grams/L). Otherwise, set to Null.
    Pattern Description: An object of type or subtype Model[Molecule], Model[Molecule, cDNA], Model[Molecule, Oligomer], Model[Molecule, Transcript], Model[Molecule, Protein], Model[Molecule, Protein, Antibody], Model[Molecule, Carbohydrate], Model[Molecule, Polymer], Model[Resin], Model[Resin, SolidPhaseSupport], Model[Lysate], Model[ProprietaryFormulation], Model[Virus], Model[Cell], Model[Cell, Mammalian], Model[Cell, Bacteria], Model[Cell, Yeast], Model[Tissue], Model[Material], or Model[Species] or Null.
    Programmatic Pattern: (ObjectP[IdentityModelTypes] | Automatic) | Null
    Index Matches to: experiment samples

    SamplePhase

    Indicates the phase of the input sample before extraction has taken place. Aqueous means that the input sample is liquid and composed only of aqueous solvents. Organic means that the input sample is liquid and composed only of organic solvents. Biphasic means that the input sample is liquid and composed of both aqueous and organic solvents that are separated into two defined layers. Unknown means that the sample phase is unknown, which will result in both Aqueous and Organic solvents being added to the input sample.
    Default Value: Automatic
    Default Calculation: Automatically set according to the PredictSamplePhase[...] function which looks at the composition of the sample's Solvent field to predict the sample's phase. If there is not enough information about the input sample for PredictSamplePhase[...] to predict the input sample's phase, a warning will be thrown. For more information, please refer to the PredictSamplePhase help file.
    Pattern Description: Aqueous, Organic, Biphasic, or Unknown.
    Programmatic Pattern: SamplePhaseP | Automatic
    Index Matches to: experiment samples

    TargetPhase

    Indicates the phase that is collected during the extraction (and carried forward to further experiments as defined by the SamplesOut field), which is the liquid layer that contains more of the dissolved TargetAnalyte after the SettlingTime has elapsed and the phases are separated.
    Figure 3.2: The TargetPhaseLayer option indicates the position (Top or Bottom) of the TargetPhase (Aqueous or Organic) in the biphasic solution, once all solvents are added.
    Default Value: Automatic
    Default Calculation: Automatically set to the phase (Organic or Aqueous) that the TargetAnalyte is more likely to be present in after extraction according to the PredictDestinationPhase[...] function. If there is not enough information for PredictDestinationPhase[...] to predict the destination phase of the target molecule, a warning will be thrown and TargetPhase will default to Aqueous. For more information, please refer to the PredictDestinationPhase help file.
    Pattern Description: Aqueous or Organic.
    Programmatic Pattern: TargetPhaseP | Automatic
    Index Matches to: experiment samples

    TargetLayer

    Indicates if the target phase is the top layer or the bottom layer of the separated solution. Note that when performing multiple rounds of extraction (NumberOfExtractions), the composition of the Aqueous and Organic layers during the first round of extraction can differ from the rest of the extraction rounds. For example, if SamplePhase->Biphasic, TargetPhase->Organic, and SelectionStrategy->Positive, the original organic layer from the input sample will be extracted and in subsequent rounds of extraction, OrganicSolvent added to the Aqueous impurity layer to extract more TargetAnalyte (the specified OrganicSolvent option can differ from the density of the original sample's organic layer). This can result in TargetLayer being different during the first round of extraction compared to the rest of the extraction rounds.
    Default Value: Automatic
    Default Calculation: Automatically calculated from the density of the input sample's aqueous and organic layers (if present in the input sample), the density of the AqueousSolvent and OrganicSolvent options (if specified), and the TargetPhase option. If density information is missing for any of these previously mentioned samples/layers, a warning will be thrown and it will be assumed that the Aqueous layer is on Top (less dense) than the Organic layer. During the calculation of this option, it is assumed that the additional molecules from the input sample will not significantly affect the densities of the aqueous and organic layers. It is also assumed that the Aqueous and Organic layers are fully separated after each round of extraction.
    Pattern Description: List of one or more Top or Bottom entries.
    Programmatic Pattern: {TargetLayerP..} | Automatic
    Index Matches to: experiment samples

    Preparation

    Indicates if this unit operation is carried out primarily robotically or manually. Manual unit operations are executed by a laboratory operator and robotic unit operations are executed by a liquid handling work cell.
    Default Value: Robotic
    Pattern Description: Robotic.
    Programmatic Pattern: Robotic

    WorkCell

    The automated workstation with a collection of integrated instruments on which this unit operation will be will be performed if Preparation -> Robotic.
    Default Value: Automatic
    Default Calculation: Automatically set to STAR if Preparation->Robotic.
    Pattern Description: STAR, bioSTAR, or microbioSTAR or Null.
    Programmatic Pattern: ((STAR | bioSTAR | microbioSTAR) | Automatic) | Null

    Phase Mixing

    SampleVolume

    The volume of the input sample that is aliquotted into the ExtractionContainer and the liquid liquid extraction is performed on.
    Default Value: Automatic
    Default Calculation: Automatically set to either half the volume of the ExtractionContainer or the Volume of the input sample, which ever is smaller, if ExtractionContainer is specified. Otherwise, is set to Null.
    Pattern Description: All or Volume or Null.
    Programmatic Pattern: ((RangeP[0*Microliter, $MaxRoboticTransferVolume] | All) | Automatic) | Null
    Index Matches to: experiment samples

    ExtractionContainer

    The container that the input sample that is aliquotted into, before the liquid liquid extraction is performed.
    Default Value: Automatic
    Default Calculation: Automatically set to Model[Container, Plate, "96-well 2mL Deep Well Plate"] if SampleVolume is set and the input sample is not in a centrifuge compatible container and Centrifugation is specified or if a non-Ambient Temperature is specified (the robotic heater/cooler units are only compatible with Plate format containers). Otherwise, if SampleVolume is set, PreferredContainer[...] is used to get a robotic compatible container that can hold the sample volume specified. Otherwise, set to Null.
    Pattern Description: Existing Container or New Container or New Container with Index or Null.
    Programmatic Pattern: (((ObjectP[Object[Container]] | _String) | ObjectP[Model[Container]] | {GreaterEqualP[1, 1], ObjectP[{Model[Container]}]}) | Automatic) | Null
    Index Matches to: experiment samples

    ExtractionContainerWell

    The well of the container that the input sample that is aliquotted into, before the liquid liquid extraction is performed.
    Default Value: Automatic
    Default Calculation: Automatically set to the first empty position in the ExtractionContainer, if specified. Otherwise, set to Null.
    Pattern Description: Any well from A1 to H12 or Null.
    Programmatic Pattern: (Alternatives @@ Flatten[AllWells[NumberOfWells -> 96]] | Automatic) | Null
    Index Matches to: experiment samples

    AqueousSolvent

    The aqueous solvent that is added to the input sample (or the impurity layer from the previous extraction round if NumberOfExtractions > 1) in order to create an organic and aqueous phase.
    Default Value: Automatic
    Default Calculation: If Aqueous solvent is required for the extraction, Model[Sample, "Milli-Q water"] is used as the AqueousSolvent. Otherwise, set to None.
    Pattern Description: An object of type or subtype Model[Sample] or Object[Sample] or a prepared sample or None.
    Programmatic Pattern: ((ObjectP[{Model[Sample], Object[Sample]}] | _String) | None) | Automatic
    Index Matches to: experiment samples

    AqueousSolventVolume

    The volume of aqueous solvent that is added and mixed with the sample during each extraction.
    Default Value: Automatic
    Default Calculation: If AqueousSolventRatio is set, AqueousSolventVolume is calculated by multiplying 1/AqueousSolventRatio with the sample volume. Otherwise, if AqueousSolvent is set, set to 20% of the volume of the sample being extracted.
    Pattern Description: Greater than or equal to 0 microliters and less than or equal to 200 milliliters or Null.
    Programmatic Pattern: (RangeP[0*Microliter, $MaxRoboticTransferVolume] | Automatic) | Null
    Index Matches to: experiment samples

    AqueousSolventRatio

    The ratio of the sample volume to the volume of aqueous solvent that is added to the sample.
    Default Value: Automatic
    Default Calculation: If AqueousSolventVolume is set, AqueousSolventRatio is calculated by dividing the sample volume by AqueousSolventVolume. Otherwise, if AqueousSolvent is set, set to 5.
    Pattern Description: Greater than 0 or Null.
    Programmatic Pattern: (GreaterP[0] | Automatic) | Null
    Index Matches to: experiment samples

    OrganicSolvent

    The organic solvent that is added to the input sample (or the impurity layer from the previous extraction round if NumberOfExtractions > 1) in order to create an organic and aqueous phase.
    Default Value: Automatic
    Default Calculation: If Organic solvent is required for the extraction and ExtractionTechnique is Pipette, Model[Sample, "Ethyl acetate, HPLC Grade"] is used as the OrganicSolvent. If Organic solvent is required for the extraction and ExtractionTechnique is PhaseSeparator, Model[Sample, "Ethyl acetate, HPLC Grade"] is used as the OrganicSolvent if it denser than the sample's Aqueous phase and the AqueousSolvent (if specified) since the phase separator will only be able to let the Organic layer pass through the hydrophobic frit if it is on the bottom. If Ethyl Acetate is not dense enough, Model[Sample, "Chloroform"] will be used. Otherwise, set to None.
    Pattern Description: An object of type or subtype Model[Sample] or Object[Sample] or a prepared sample or None.
    Programmatic Pattern: ((ObjectP[{Model[Sample], Object[Sample]}] | _String) | None) | Automatic
    Index Matches to: experiment samples

    OrganicSolventVolume

    The volume of organic solvent that is added and mixed with the sample during each extraction.
    Default Value: Automatic
    Default Calculation: If OrganicSolventRatio is set, OrganicSolventVolume is calculated by multiplying 1/OrganicSolventRatio with the sample volume. Otherwise, if OrganicSolvent is set, set to 20% of the volume of the sample being extracted.
    Pattern Description: Greater than or equal to 0 microliters and less than or equal to 200 milliliters or Null.
    Programmatic Pattern: (RangeP[0*Microliter, $MaxRoboticTransferVolume] | Automatic) | Null
    Index Matches to: experiment samples

    OrganicSolventRatio

    The ratio of the sample volume to the volume of organic solvent that is added to the sample.
    Default Value: Automatic
    Default Calculation: If OrganicSolventVolume is set, OrganicSolventRatio is calculated by dividing the sample volume by OrganicSolventVolume. Otherwise, if OrganicSolvent is set, set to 5.
    Pattern Description: Greater than 0 or Null.
    Programmatic Pattern: (GreaterP[0] | Automatic) | Null
    Index Matches to: experiment samples

    SolventAdditions

    For each extraction round, the solvent(s) that are added to the sample in order to create a biphasic solution.
    Default Value: Automatic
    Default Calculation: Aqueous solvent is automatically added if the starting sample of each extraction round is of Organic or Unknown phase. Organic solvent is automatically added if the starting sample of each extraction round is of Aqueous or Unknown phase. If the sample is already Biphasic, the no solvent is added. Note that the phase of the starting sample in extraction rounds 2 and above is dependent on the TargetPhase and SelectionStrategy options.
    Pattern Description: List of one or more Multiple Solvents or No Solvent or Single Solvent entries.
    Programmatic Pattern: {((ObjectP[{Model[Sample], Object[Sample]}] | _String) | {(ObjectP[{Model[Sample], Object[Sample]}] | _String)..} | None)..} | Automatic
    Index Matches to: experiment samples

    Demulsifier

    The solution that is added to the sample mixture in order to help promote complete phase separation and avoid emulsions.
    Default Value: Automatic
    Default Calculation: If DemulsifierAdditions is specified, automatically set to the demulsifier specified in DemulsifierAdditions. Otherwise, automatically set to Model[Sample, StockSolution, "5M Sodium Chloride"] if DemulsifierAmount is specified. Otherwise, set to Null.
    Pattern Description: An object of type or subtype Model[Sample] or Object[Sample] or a prepared sample or None.
    Programmatic Pattern: ((ObjectP[{Model[Sample], Object[Sample]}] | _String) | None) | Automatic
    Index Matches to: experiment samples

    DemulsifierAmount

    The solution that is added to the sample mixture in order to help promote complete phase separation and avoid emulsions.
    Default Value: Automatic
    Default Calculation: Automatically set to 10% of the sample volume if Demulsifier or DemulsifierAdditions is specified. Otherwise, set to Null.
    Pattern Description: Greater than or equal to 0 milliliters and less than or equal to 200 milliliters or None or Null.
    Programmatic Pattern: ((RangeP[0*Milliliter, $MaxRoboticTransferVolume] | None) | Automatic) | Null
    Index Matches to: experiment samples

    DemulsifierAdditions

    For each extraction round, the Demulsifier that is added to the sample mixture to help promote complete phase separation and avoid emulsions.
    Default Value: Automatic
    Default Calculation: If Demulsifier is not specified, DemulsifierAdditions is set to None. If NumberOfExtractions is set to 1, Demulsifier will only be added during the first extraction round. If NumberOfExtractions is greater than 1 and the sample's Organic phase will be used subsequent extraction rounds (TargetPhase->Aqueous and ExtractionTechnique->Positive OR TargetPhase->Organic and ExtractionTechnique->Negative), Demulsifier will be added during all extraction rounds since the Demulsifier (usually a salt solution which is soluble in the Aqueous layer) will be removed along with the Aqueous layer during the extraction and thus will need to be added before each extraction round. Otherwise, Demulsifier is added to only the first extraction round since the sample's Aqueous phase will be used in subsequent extraction rounds.
    Pattern Description: List of one or more Demulsifier or None entries.
    Programmatic Pattern: {((ObjectP[{Model[Sample], Object[Sample]}] | _String) | None)..} | Automatic
    Index Matches to: experiment samples

    Temperature

    The set temperature of the incubation device that holds the extraction container during solvent/demulsifier addition, mixing, and settling.
    Default Value: Ambient
    Pattern Description: Ambient or greater than or equal to 0 degrees Celsius and less than or equal to 110 degrees Celsius.
    Programmatic Pattern: RangeP[$MinRoboticIncubationTemperature, $MaxRoboticIncubationTemperature] | Ambient
    Index Matches to: experiment samples

    NumberOfExtractions

    The number of times that the extraction is performed using the specified extraction parameters using the previous extraction round's impurity layer (after the TargetPhase has been extracted) as the input to subsequent rounds of extraction.
    Figure 3.3: The NumberOfExtractions option indicates the number of times that Aqueous and/or Organic solvent is added to the Target or Impurity layer (depending on if SelectionStrategy is set to Negative or Positive). Note that regardless of the SelectionStrategy or NumberOfExtractions, all impurity layers and target layers will be pooled together such that there is one impurity sample and one target sample at the end of the liquid liquid extraction.
    Default Value: 3
    Pattern Description: Greater than or equal to 1 and less than or equal to 10 in increments of 1.
    Programmatic Pattern: RangeP[1, 10, 1]
    Index Matches to: experiment samples

    ExtractionMixType

    The style of motion used to mix the sample mixture following the addition of the AqueousSolvent/OrganicSolvent and Demulsifier (if specified).
    Default Value: Automatic
    Default Calculation: Automatically set to Shake if ExtractionMixTime is specified. Otherwise, set to Pipette.
    Pattern Description: Pipette or Shake or Null.
    Programmatic Pattern: (RoboticMixTypeP | Automatic) | Null
    Index Matches to: experiment samples

    ExtractionMixTime

    The duration for which the sample, AqueousSolvent/OrganicSolvent, and Demulsifier (if specified) are mixed.
    Default Value: Automatic
    Default Calculation: Automatically set to 30 Second if ExtractionMixType is set to Shake. Otherwise, set to Null.
    Pattern Description: Greater than or equal to 0 seconds and less than or equal to 72 hours or Null.
    Programmatic Pattern: (RangeP[0*Second, $MaxExperimentTime] | Automatic) | Null
    Index Matches to: experiment samples

    ExtractionMixRate

    The frequency of rotation the mixing instrument uses to mechanically incorporate the sample, AqueousSolvent/OrganicSolvent, and demulsifier (if specified).
    Default Value: Automatic
    Default Calculation: Automatically set to 300 RPM if ExtractionMixType is set to Shake. Otherwise, set to Null.
    Pattern Description: Greater than or equal to 30 revolutions per minute and less than or equal to 2500 revolutions per minute or Null.
    Programmatic Pattern: (RangeP[$MinRoboticMixRate, $MaxRoboticMixRate] | Automatic) | Null
    Index Matches to: experiment samples

    NumberOfExtractionMixes

    The number of times the sample, AqueousSolvent/OrganicSolvent, and demulsifier (if specified) are mixed when ExtractionMixType is set to Pipette.
    Default Value: Automatic
    Default Calculation: Automatically set to 10 when ExtractionMixType is set to Pipette. Otherwise, set to Null.
    Pattern Description: Greater than or equal to 1 and less than or equal to 100 in increments of 1 or Null.
    Programmatic Pattern: (RangeP[1, $MaxNumberOfMixes, 1] | Automatic) | Null
    Index Matches to: experiment samples

    ExtractionMixVolume

    The volume of sample, AqueousSolvent/OrganicSolvent, and demulsifier (if specified) that is mixed when ExtractionMixType is set to Pipette.
    Default Value: Automatic
    Default Calculation: Automatically set to the lesser of 1/2 of the volume of the sample plus any additional components (SampleVolume, AqueousSolventVolume (if specified), OrganicSolventVolume (if specified), and DemulsifierAmount (if specified)) and 970 Microliter (the maximum amount of volume that can be transferred in a single pipetting step on the liquid handling robot) if ExtractionMixType is set to Pipette. Otherwise, set to Null.
    Pattern Description: Greater than or equal to 0 milliliters and less than or equal to 970 microliters or Null.
    Programmatic Pattern: (RangeP[0*Milliliter, $MaxRoboticSingleTransferVolume] | Automatic) | Null
    Index Matches to: experiment samples

    Settling

    SettlingTime

    The duration for which the sample is allowed to settle and the organic/aqueous phases separate. This is performed after the AqueousSolvent/OrganicSolvent and Demulsifier (if specified) are added and optionally mixed. If ExtractionTechnique is set to PhaseSeparator, the settling time starts once the sample is loaded into the phase separator (the amount of time that we wait for the organic layer to drain through the phase separator's hydrophobic frit).
    Default Value: 1 minute
    Pattern Description: Greater than or equal to 0 seconds and less than or equal to 72 hours or Null.
    Programmatic Pattern: RangeP[0*Second, $MaxExperimentTime] | Null
    Index Matches to: experiment samples

    Centrifuge

    Indicates if the sample is centrifuged to help separate the aqueous and organic layers, after the addition of solvent/demulsifier, mixing, and setting time has elapsed.
    Default Value: Automatic
    Default Calculation: Automatically set to True any of the other centrifuge options are specified (CentrifugeInstrument, CentrifugeIntensity, CentrifugeTime). Also automatically set to True if ExtractionTechnique -> Pipette and the samples are in a centrifuge compatible container (the Footprint of the container is set to Plate). Otherwise, set to False.
    Pattern Description: True or False.
    Programmatic Pattern: BooleanP | Automatic
    Index Matches to: experiment samples

    CentrifugeInstrument

    The centrifuge that is used to spin the samples to help separate the aqueous and organic layers, after the addition of solvent/demulsifier, mixing, and setting time has elapsed.
    Default Value: Automatic
    Default Calculation: Automatically set to the integrated centrifuge model that is available in the WorkCell, if the Centrifuge option is set to True.
    Pattern Description: An object of type or subtype Model[Instrument, Centrifuge] or Object[Instrument, Centrifuge] or Null.
    Programmatic Pattern: (ObjectP[{Model[Instrument, Centrifuge], Object[Instrument, Centrifuge]}] | Automatic) | Null
    Index Matches to: experiment samples

    CentrifugeIntensity

    The rotational speed or the force that is applied to the samples via centrifugation to help separate the aqueous and organic layers, after the addition of solvent/demulsifier, mixing, and setting time has elapsed.
    Default Value: Automatic
    Default Calculation: Automatically set to the lesser of the MaxIntensity of the centrifuge model and the MaxCentrifugationForce of the plate model, if centrifugation is specified.
    Pattern Description: Greater than 0 revolutions per minute or greater than 0 standard accelerations due to gravity on the surface of the earth or Null.
    Programmatic Pattern: ((GreaterP[0*RPM] | GreaterP[0*GravitationalAcceleration]) | Automatic) | Null
    Index Matches to: experiment samples

    CentrifugeTime

    The amount of time that the samples are centrifuged to help separate the aqueous and organic layers, after the addition of solvent/demulsifier, mixing, and setting time has elapsed.
    Default Value: Automatic
    Default Calculation: Automatically set to 2 Minute, if centrifugation is specified.
    Pattern Description: Greater than or equal to 0 seconds and less than or equal to 72 hours or Null.
    Programmatic Pattern: (RangeP[0*Second, $MaxExperimentTime] | Automatic) | Null
    Index Matches to: experiment samples

    Collection

    ExtractionBoundaryVolume

    For each extraction round, the volume of the target phase that is either overaspirated via Pipette when IncludeBoundary is set to True (by aspirating the boundary layer along with the TargetPhase and therefore potentially collecting a small amount of the unwanted phase) or underaspirated via Pipette when IncludeBoundary is set to False (by not collecting all of the target phase and therefore reducing the likelihood of collecting any of the unwanted phase). This option only applies if ExtractionTechnique -> Pipette.
    Default Value: Automatic
    Default Calculation: Automatically set the smaller of 10% of the predicted volume of the ExtractionTransferLayer or the volume that corresponds with a 5 Millimeter tall cross-section of the ExtractionContainer at the position of the boundary between aqueous and organic layers if ExtractionTransferLayer -> Top or at the bottom of the container if ExtractionTransferLayer -> Bottom. If ExtractionTechnique -> PhaseSeparator, set to Null.
    Pattern Description: List of one or more greater than or equal to 0 milliliters and less than or equal to 200 milliliters entries or Null.
    Programmatic Pattern: ({RangeP[0*Milliliter, $MaxRoboticTransferVolume]..} | Automatic) | Null
    Index Matches to: experiment samples

    ExtractionTransferLayer

    Indicates whether the top or bottom layer is transferred from the source sample after the organic and aqueous phases are separated. If the TargetLayer matches ExtractionTransferLayer, the sample that is transferred out is the target phase. Otherwise, if TargetLayer doesn't match ExtractionTransferLayer, the sample that remains in the container after the transfer is the target phase.
    Figure 3.4: The ExtractionTransferLayer indicates the layer (Top or Bottom) that should be removed from the biphasic solution if ExtractionTechnique->Pipette (does not apply to phase separators). The boundary between the layers will be aspirate along with the ExtractionTransferLayer if IncludeBoundary -> True. Note that if ExtractionTransferLayer matches TargetPhaseLayer (the TargetPhase is being aspirated), setting IncludeBoundary->True will maximize yield, otherwise, setting IncludeBoundary->False will maximize purity. The inverse is true if ExtractionTransferLayer does not match TargetPhaseLayer (the Impurity phase is being aspirated).
    Default Value: Automatic
    Default Calculation: Automatically set to Top if ExtractionTechnique->Pipette. Otherwise, set to Null.
    Pattern Description: List of one or more Top or Bottom entries or Null.
    Programmatic Pattern: ({(Top | Bottom)..} | Automatic) | Null
    Index Matches to: experiment samples

    TargetContainerOut

    The container that the separated target layer is transferred into (either via Pipette or PhaseSeparator) after the organic and aqueous phases are separated.
    Default Value: Automatic
    Default Calculation: Automatically set to Model[Container, Plate, "96-well 2mL Deep Well Plate"] if TargetPhase -> Organic and ExtractionTechnique -> PhaseSeparator (since the organic layer will flow through the phase separator's hydrophobic frit, TargetContainerOut will be used as the collection container for the phase separator). Otherwise, automatically set to a robotic compatible container that can hold the volume of the target layer via PreferredContainer[...].
    Pattern Description: Existing Container or New Container or New Container with Index.
    Programmatic Pattern: ((ObjectP[Object[Container]] | _String) | ObjectP[Model[Container]] | {GreaterEqualP[1, 1], ObjectP[{Model[Container]}]}) | Automatic
    Index Matches to: experiment samples

    TargetContainerOutWell

    The well of the container that the separated target layer is transferred into (either via Pipette or PhaseSeparator) after the organic and aqueous phases are separated.
    Default Value: Automatic
    Default Calculation: Automatically set the first empty well in TargetContainerOut.
    Pattern Description: Any well from A1 to H12.
    Programmatic Pattern: Alternatives @@ Flatten[AllWells[NumberOfWells -> 96]] | Automatic
    Index Matches to: experiment samples

    ImpurityContainerOut

    The container that the separated impurity layer is transferred into (either via Pipette or PhaseSeparator) after the organic and aqueous phases are separated.
    Default Value: Automatic
    Default Calculation: Automatically set to Model[Container, Plate, "96-well 2mL Deep Well Plate"] if TargetPhase -> Aqueous and ExtractionTechnique -> PhaseSeparator (since the organic impurity layer will flow through the phase separator's hydrophobic frit, ImpurityContainerOut will be used as the collection container for the phase separator). Otherwise, automatically set to a robotic compatible container that can hold the volume of the target layer via PreferredContainer[...].
    Pattern Description: Existing Container or New Container or New Container with Index.
    Programmatic Pattern: ((ObjectP[Object[Container]] | _String) | ObjectP[Model[Container]] | {GreaterEqualP[1, 1], ObjectP[{Model[Container]}]}) | Automatic
    Index Matches to: experiment samples

    ImpurityContainerOutWell

    The well of the container that the separated impurity layer is transferred into (either via Pipette or PhaseSeparator) after the organic and aqueous phases are separated.
    Default Value: Automatic
    Default Calculation: Automatically set the first empty well in ImpurityContainerOut.
    Pattern Description: Any well from A1 to H12.
    Programmatic Pattern: Alternatives @@ Flatten[AllWells[NumberOfWells -> 96]] | Automatic
    Index Matches to: experiment samples

    TargetStorageCondition

    The condition under which the target sample is stored after the protocol is completed. If left unset, the target sample will be stored under the same condition as the source sample that it originates from.
    Default Value: Null
    Pattern Description: Storage Object or Storage Type or Null.
    Programmatic Pattern: ((SampleStorageTypeP | Disposal) | ObjectP[Model[StorageCondition]]) | Null
    Index Matches to: experiment samples

    ImpurityStorageCondition

    The conditions under which the waste layer samples will be stored after the protocol is completed. If left unset, the waste sample will be stored under the same condition as the source sample that it originates from.
    Default Value: Null
    Pattern Description: Storage Object or Storage Type or Null.
    Programmatic Pattern: ((SampleStorageTypeP | Disposal) | ObjectP[Model[StorageCondition]]) | Null
    Index Matches to: experiment samples

    Post Experiment

    SamplesInStorageCondition

    The non-default conditions under which the SamplesIn of this experiment should be stored after the protocol is completed. If left unset, SamplesIn will be stored according to their current StorageCondition.
    Default Value: Null
    Pattern Description: {AmbientStorage, EnclosedAmbientStorage, Refrigerator, Freezer, DeepFreezer, CryogenicStorage, YeastIncubation, YeastShakingIncubation, BacterialIncubation, BacterialShakingIncubation, MammalianIncubation, ViralIncubation, CrystalIncubation, AcceleratedTesting, IntermediateTesting, LongTermTesting, UVVisLightTesting} or Disposal or Null.
    Programmatic Pattern: (Alternatives[SampleStorageTypeP | Disposal]) | Null
    Index Matches to: experiment samples

    Model Input

    PreparedModelContainer

    Indicates the container in which a Model[Sample] specified as input to the experiment function will be prepared.
    Default Value: Automatic
    Default Calculation: If PreparedModelAmount is set to All and when the input model has a product associated with both Amount and DefaultContainerModel populated, automatically set to the DefaultContainerModel value in the product. Otherwise set to Model[Container, Vessel, "2mL Tube"].
    Pattern Description: An object of type or subtype Model[Container] or Null.
    Programmatic Pattern: (ObjectP[Model[Container]] | Automatic) | Null
    Index Matches to: experiment samples

    PreparedModelAmount

    Indicates the amount of a Model[Sample] specified as input to the experiment function that will be prepared in the PreparedModelContainer. When set to All and the input model sample is not preparable, the entire amount of the input model sample that comes from one of the Products is prepared. The selected product must have both Amount and DefaultContainerModel populated, and it must not be a KitProduct. When set to All and the input model is preparable such as water, 1 Milliliter of the input model sample is prepared.
    Default Value: Automatic
    Default Calculation: Automatically set to 1 Milliliter.
    Pattern Description: All or Count or Count or Mass or Volume or Null.
    Programmatic Pattern: ((RangeP[1*Microliter, 20*Liter] | RangeP[1*Milligram, 20*Kilogram] | GreaterP[0*Unit, 1*Unit] | GreaterP[0., 1.] | All) | Automatic) | Null
    Index Matches to: experiment samples

Sample Prep Options

    Sample Preparation

    PreparatoryUnitOperations

    Specifies a sequence of transferring, aliquoting, consolidating, or mixing of new or existing samples before the main experiment. These prepared samples can be used in the main experiment by referencing their defined name. For more information, please reference the documentation for ExperimentSamplePreparation.
    Default Value: Null
    Pattern Description: List of one or more unit Operation ManualSamplePreparation or RoboticSamplePreparation or unit Operation must match SamplePreparationP entries or Null.
    Programmatic Pattern: {((ManualSamplePreparationMethodP | RoboticSamplePreparationMethodP) | SamplePreparationP)..} | Null

Protocol Options

    Organizational Information

    Template

    A template protocol whose methodology should be reproduced in running this experiment. Option values will be inherited from the template protocol, but can be individually overridden by directly specifying values for those options to this Experiment function.
    Default Value: Null
    Pattern Description: An object of type or subtype Object[Protocol] or an object of type or subtype of Object[Protocol] with UnresolvedOptions, ResolvedOptions specified or Null.
    Programmatic Pattern: (ObjectP[Object[Protocol]] | FieldReferenceP[Object[Protocol], {UnresolvedOptions, ResolvedOptions}]) | Null

    Name

    A object name which should be used to refer to the output object in lieu of an automatically generated ID number.
    Default Value: Null
    Pattern Description: A string or Null.
    Programmatic Pattern: _String | Null

    Post Experiment

    MeasureWeight

    Indicates if any solid samples that are modified in the course of the experiment should have their weights measured and updated after running the experiment. Please note that public samples are weighed regardless of the value of this option.
    Default Value: Automatic
    Pattern Description: True or False or Null.
    Programmatic Pattern: (BooleanP | Automatic) | Null

    MeasureVolume

    Indicates if any liquid samples that are modified in the course of the experiment should have their volumes measured and updated after running the experiment. Please note that public samples are volume measured regardless of the value of this option.
    Default Value: Automatic
    Pattern Description: True or False or Null.
    Programmatic Pattern: (BooleanP | Automatic) | Null

    ImageSample

    Indicates if any samples that are modified in the course of the experiment should be freshly imaged after running the experiment. Please note that public samples are imaged regardless of the value of this option.
    Default Value: Automatic
    Pattern Description: True or False or Null.
    Programmatic Pattern: (BooleanP | Automatic) | Null

Example Calls

    Basic Examples

    Basic liquid liquid extraction with an aqueous sample:
    Specify the OrganicSolvent to be used in the extraction:
    Specify that the sample will be centrifuged to promote phase separation, after the solvents are added:
    Basic liquid liquid extraction with an aqueous sample with ExtractionTechnique->PhaseSeparator. Phase separators can only be used when the density of the organic layer is greater than the density of the aqueous layer since the hydrophobic frit of phase separator stops the aqueous layer from draining into the collection plate (and if the aqueous layer is on the bottom, no sample will pass through the hydrophobic frit). If compatible with your sample/solvents, it is recommended to use a PhaseSeparator over a Pipette to separate the aqueous and organic layers:
    Basic liquid liquid extraction with an aqueous sample with ExtractionTechnique->Pipette:
    Specify multiple extraction rounds (3 extraction rounds are recommended):
    Maximize yield by setting SelectionStrategy -> Positive (see the SelectionStrategy option for more information):
    Maximize purity by setting SelectionStrategy -> Negative (see the SelectionStrategy option for more information):

    Phase Prediction

    The SamplePhase (Aqueous, Organic, Biphasic, or Unknown) of the input sample can be specified. It is automatically predicted from the sample's Solvent and Composition field via the PredictSamplePhase function (refer to PredictSamplePhase documentation for more information):
    A TargetAnalyte will automatically be calculated from the Analytes field of the sample. The TargetAnalyte can also be manually specified (to set the Analytes field in the input sample, use the function DefineAnalytes):
    The TargetAnalyte will be used to automatically calculate the TargetPhase (the phase in which the TargetAnalyte is more concentrated) via the function PredictDestinationPhase. Please refer to the documentation of PredictDestinationPhase for more information. The TargetPhase can also be manually specified:
    The densities of the aqueous / organic phases will be used to determine if the TargetPhase is on the bottom or on the top. If the density of either of the phases is unknown, a warning will be thrown. The TargetLayer option (which indicates if the TargetPhase is on bottom or on top) can also be manually specified:

    Pipette Specific Options

    When performing a Liquid Liquid Extraction with a pipette, the ExtractionTransferLayer specifies the layer that should be aspirated off using a pipette. By default, the ExtractionTransferLayer is set to Top since aspirating off the Bottom layer risks contamination since the pipette would have to go through the Top layer to aspirate off the Bottom layer:
    When performing a Liquid Liquid Extraction with a pipette, ExtractionBoundaryVolume is the volume of the target phase that is either overaspirated via Pipette when IncludeBoundary is set to True (by aspirating the boundary layer along with the TargetPhase and therefore potentially collecting a small amount of the unwanted phase) or underaspirated via Pipette when IncludeBoundary is set to False:

    Selection Strategy

    SelectionStrategy -> Positive indicates that additional rounds of extraction are performed on the impurity phase. Positive selection is used when the goal is to extract the maximum amount of TargetAnalyte from the impurity phase (maximizing yield). Please consult the SelectionStrategy option diagram for more information:
    SelectionStrategy -> Negative indicates that additional rounds of extraction are performed on the target phase. Negative selection is used when the goal is to remove impurities that may still exist in the TargetPhase (maximizing purity). Please consult the SelectionStrategy option diagram for more information:

Preferred Input Containers

    SBS Format Plates
    2mL and 50mL Tubes

Warnings and Errors

    Messages  (17)

    ConflictingCentrifugeContainerParametersForLLE  (1)

    Return an error if there are conflicting centrifugation options specified:

    ConflictingCentrifugeParametersForLLE  (1)

    Return an error if there are conflicting mix options specified:

    ConflictingMixParametersForLLE  (1)

    Return an error if there are conflicting mix options specified:

    ConflictingTemperaturesForLLE  (1)

    Return an error if there are conflicting temperature options specified:

    DemulsifierSpecifiedConflict  (1)

    Return an error if there is a conflict between the DemulsifierAdditions and Demulsifier options:

    ExtractionOptionMismatch  (1)

    Return an error if there are conflicting extraction options specified:

    InvalidExtractionRoundLengths  (1)

    Return an error if the length of the SolventAdditions option does not match the NumberOfExtractions option:

    InvalidSolventAdditions  (1)

    Return an error if the Solvents specified will be inadequate to create a biphasic solution for liquid liquid extraction:

    ObjectDoesNotExist  (6)

    Do NOT throw a message if we have a simulated container but a simulation is specified that indicates that it is simulated:

    Do NOT throw a message if we have a simulated sample but a simulation is specified that indicates that it is simulated:

    Throw a message if we have a container that does not exist (ID form):

    Throw a message if we have a container that does not exist (name form):

    Throw a message if we have a sample that does not exist (ID form):

    Throw a message if we have a sample that does not exist (name form):

    OrganicSolventDensityPhaseSeparator  (1)

    Return an error if the user requests to use a PhaseSeparator but the Organic layer will be on top of the Aqueous layer and therefore will not be able to pass through the hydrophobic frit:

    SolventAdditionsMismatch  (1)

    Return an error if the Solvents specified will be inadequate to create a biphasic solution for liquid liquid extraction:

    WeakTargetAnalytePhaseAffinity  (1)

    Throw a warning if the target analytes specified have a weak affinity (according to the XLogP algorithm or experimentally measured LogP) to both the Aqueous and Organic phase and a TargetPhase isn't specified:

Possible Issues

    Lack of Phase Separation

    If inappropriate AqueousSolvent / OrganicSolvent options are specified, the sample can fail to separate into a biphasic solution. Ethyl Acetate and Chloroform are used by default as Organic Solvents and Water is used as default as an Aqueous Solvent.

    Phase Separator Failure

    The Phase Separator allows the Organic phase of the sample to flow through the hydrophobic frit via gravity, into a collection container that is stacked on the bottom of the phase separator. If the Organic phase of the sample is less dense than the Aqueous phase of the sample, the Aqueous phase will be on the bottom and the Organic phase will not be able to pass through the hydrophobic frit. The LLE function checks that the density of the AqueousSolvent is less than the density of the OrganicSolvent, but this assumes that the other components in the sample will not affect the density of these phases. It is recommended that when using a Phase Separator that the OrganicSolvent should be much denser than the AqueousSolvent. By default, Chloroform is used as the OrganicSolvent when using a Phase Separator if an OrganicSolvent is not already provided.

    Emulsions

    Depending on the sample and solvents, an emulsion can form -- this is when one of the phases breaks up into droplets and starts to spread throughout the other phase. This can occur if the solution contains surfactant-like compounds which will be soluble in both the Aqueous and Organic phase (the hydrophobic part of the surfactant wants to stay close to the Organic phase but the hydrophilic part of the surfactant wants to stay close to the Aqueous phase, causing small droplets to form throughout the solution). To break an emulsion, (1) the Centrifuge option can be used to physically separate the two phases before extraction, (2) the Demulsifier option can "salt out" since the addition of salt increases the ionic strength of the aqueous layer and forces the surfactant-like compounds to separate into either the aqueous or the organic layer (instead of partial separation into both), (3) SolidPhaseExtraction (SPE) can be used instead of LLE so that emulsions don't affect the extraction process.
Last modified on Fri 5 Sep 2025 17:19:46

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