Agilent 1200 HPLC System: Fraction Collector

Commands and Tips for Third-Party Devices
Agilent 1200 HPLC System: Fraction Collector

This topic describes the major fraction collection commands and properties and how they are used in an application.

General commands and properties for fraction collection

Property

Description/Setting

FractionationMode

Determines the mode for fraction collection. The following modes are available:

ByTime: Select to collect fractions based on time intervals.

ByVolume: Select to collect fractions based on a specified volume.

ByPeak: Select to collect fractions based on peaks in a chromatographic signal.

MaxTubeNumberPerSample

Specifies the maximum number of fractions to be collected during a single analysis.

To collect fractions by time

In time-based fractionation, each fraction is collected for a specified time period. The following commands and properties are used for the time-based mode:

Property

Description/Setting

FractionDuration

Specifies the length of a fraction.

Note: In addition, you have to set the related property FractionMode to the value ByTime.

Example:

To change to the next tube every 10,000 seconds, the sample PGM must include the following commands:

FractionationMode = By_Time

FractionDuration = 10.000 [s]

To collect fractions by volume

The following commands and properties are used to perform volume-based fractionation:

Property

Description

FractionVolume

Specifies the volume of the fraction.

Note: In addition, you have to set the related property FractionMode to the value ByVolume.

Example:

To change to the next tube after delivery of every 10,000 ml, the sample PGM must contain the following commands:

FractionationMode = By_Volume

FractionVolume = 10.000 [ml]

To collect fractions by peak

In peak-based fractionation, the signal for the first channel of the respective detector (i.e., UV_VIS_1 and FL_1) is evaluated. In this case, the length of a fraction is determined by the peak start and peak end. The following commands and properties are used to set the peak detection mode of the detector and to specify the peak start and end triggers:

Property	Description
FractionTimeout	Specifies the maximum length of a fraction. If a peak start but no peak end was detected, the fraction will end after expiration of the specified time.
FldPeakDetectionMode/ UvdPeakDetectionMode	Specifies the peak detection mode for the FLD and/or UV detector. ThresholdBased – Select to collect fractions based on a specified threshold value only. Values for DetectionUpSlope and DetectionDownSlope are ignored. SlopeBased – Select to collect fractions based on the up and down slope of the detector signal. The value for DetectionThreshold is ignored. Combined – Select to collect fractions based both on the slope and threshold values. Fraction collection starts if the detector signal exceeds the up slope or threshold value. Fraction collection ends if the detector signal falls below either the threshold or the down slope value. Off – Select if you do not want to detect peaks using the respective detector (UV or FLD).
FldPeakDetectionUpSlope/ UvdPeakDetectionUpSlope	Enter a value for the up slope to determine a peak start. When the slope of the peak rises above the value entered for up slope, peak start is triggered and fraction collection begins.
FldPeakDetectionDownSlope/ UvdPeakDetectionDownSlope	Enter a value for the down slope to determine a peak end. When the slope of the peak falls below the value entered for down slope, peak end is triggered and fraction collection ends.
FldPeakDetectionThreshold/ UvdPeakDetectionThreshold	Specifies a threshold to determine peak start/end. When the detector signal rises above the specified threshold value, peak start is triggered. When the detector signal falls below the threshold value, peak end is triggered.
FldPeakDetectionUpperThreshold/ UvdPeakDetectionUpperThreshold	Specifies an upper threshold to prevent false fraction collection triggering in case of detector noise. Set the upper threshold value below the limit where false triggering might occur. While the detector signal exceeds the upper threshold, all other peak detection settings will be ignored until the signal falls down again below the upper threshold.
TriggerCombination	When using more than one detector, you can specify how fraction collection triggers defined for the used detectors are combined. Any – Fraction collection starts when the first detector detects a peak start and ends after all detectors detected a peak end. All – Fraction collection starts when all detectors detected a peak start and ends when any of the detectors detected a peak end.

To define the start location for fraction collection

Property	Description
CollectPosition	Shows the position used for the current fraction.
TubePosition	Specifies the tube position of the first fraction to be collected. If no position is specified, i.e., if AutoIncrement is selected, the first position of the next fraction is calculated using the last position of the previous fraction. The behavior depends on the selected meandering scheme. Examples: In the PGM, define a fixed value for TubePosition, if you want to collect fractions into the same vials during a sequence. The sample PGM should contain the following command: TubePosition = <desired position>. In the PGM, set TubePosition to AutoIncrement and specify the position of the first sample to be fractionated on the panel, if you want to perform several subsequent analyses which contain different numbers of fractions. The sample PGM should contain the following command: TubePosition = AutoIncrement. Related properties: MeanderingScheme, CollectPosition.
TubeVolumeReset	Resets the specified collection volume to zero for all tubes.

To specify the needle height

Use the following properties to specify how deep the needle descends into the sample container.

Property	Description
DiverterValveRemainsOpen	Specifies whether the diverter valve is opened or closed during needle movement. Note: The positions of the sample container must be neighbored in either vertical or horizontal direction and the sample container to be used must be a well plate.
DropOnSurface	(Only required for analytical scale fraction collector) The needle moves down to the well/vial bottom until the forming droplet contacts the surface of the liquid. As the vial/well is filled with the collected liquid, the needle moves upwards while the droplet stays in contact with the surface of the rising liquid. This procedure prevents forming of air bubbles and ensures that at the end of the fraction, no droplet remains on the needle tip.
VialsCapped	(Only provided for analytical scale fraction collector) Specifies whether capped vials are used or not. When set to Yes, the needle moves down for the specified distance to pierce the septum of the vial before the diverter valve opens. Related properties: NeedleDepth, NeedleAboveLocation, DiverterValveRemainsOpen.
NeedleAboveLocation	Specifies the distance in [mm] between the bottom of the sample container (well plate, deep well plate, or vial), and the tip of the needle. The distance can be set in increments of 0.1mm. Note: This property is only relevant, if the related property VialsCapped is set to No.
NeedleDepth	Specifies how deep the needle moves into the vial. Note: This property is only relevant if the related property VialsCapped is set to Yes.

To specify the collection scheme

The collection scheme describes the path that is followed by the needle when collecting fractions.

Property	Description
MeanderingScheme	Specifies the collection scheme. The following schemes are possible:
	RowByRow (default) This scheme is suitable for all supported tray types, except for 60 and 215 position trays.
	RowByRow60: Same as RowbyRow but only for trays with 60 positions.
	RowByRow215: Same as RowbyRow but only for trays with 215 positions.
	RowByRowMeandering The needle moves row by row, following a meander-shaped path. This scheme is suitable for all supported tray types, except for 60 and 215 position trays.
	RowByRowMeandering60: Same as RowByRowMeandering but only for trays with 60 positions.
	RowByRowMeandering215: Same as RowByRowMeandering but only for trays with 215 positions.
	ColumnByColumn This scheme is suitable for all tray types that are supported by Chromeleon.
	ColumnByColumnMeandering The needle moves column by column, following a meander-shaped path. This scheme is suitable for all supported tray types, except for 60 and 215 position trays.
	ColumnByColumnMeandering60: Same as ColumnByColumnMeandering but only for trays with 60 positions.
	ColumnByColumnMeandering215: Same as ColumnByColumnMeandering but only for trays with 215 positions.
	StayAtRinseStation: No movement, the needle stays in the rinse station.

Tip:

The numbering of positions on a tray varies depending on the selected tray and meandering scheme. To identify the numbering scheme of the used tray and to ensure that the correct position numbers are used for the TubePosition property, perform a test run and then check the position numbering in the Fraction Tray View. To display the fraction tray, click the (Show Fraction Tray) icon in the Chromeleon Toolbar.

To perform delay time or dead volume calibration

You can use the commands/properties below to calibrate the fraction collector and thus determine the dead volume or delay time that exists between the detectors and fractionation valve.

After successful calibration, calculations of the dead volume between valve and needle tip, valve switching or needle movement delay times are performed automatically by the fraction collector.

For detailed information on how to perform delay time/dead volume calibration, also refer to the operating manual of the fraction collector.

Property	Description
FC_DelaySensor	Records the signal of the delay sensor as a third detector signal at a wavelength of 654 nm. Click the plus sign beside the name to display the following commands and properties:
	AcqOn/AcqOff	AcqOn starts data acquisition. AcqOff terminates data acquisition.
	Average	Averages all measured values over the step interval. Default: On. Off records only the last point of each interval.
	Delta	Reports the signal slope, that is, the difference between the value and the value 1 second ago. This is useful for Triggers.
	MaxAutoStep	Sets the maximum step rate for Auto Step Mode. Range: 0.1 - 5.1 s. Default: 5.1 s.
	Retention	Reports the retention time for the signal.
	Signal	Reports the signal value. It has the following commands: Value (current signal value), UpperLimit, and LowerLimit. If the current signal value is outside these limits, a warning appears in the Audit Trail.
	Step	Sets the step for data acquisition. Auto selects the best step dynamically.
FldDelayTime	The time that a substance needs to travel from the first detector to the fraction collection valve. The delay time depends on the current flow and the FldDeadVolume. In peak-based fractionation, the fraction collector switches the fractionation valve when a peak has been detected by the detector plus the delay time. Tip: When you have specified a delay time, the flow rate must be kept constant during the run. Otherwise, the calculated delay times and delay offsets between the detector and the fraction collector can no longer be used.
FldDeadVolume	Indicates the volume between the FLD detector's flow cell and the fraction collection valve.
UvdDelayTime	Same as FldDelayTime but for UVD detector.
UvdDeadVolume	Same as FldDelayVolume but for UVD detector.

Additional tips for fraction collection

For a correct calculation of the delay time and/or dead volume and the correct display of overlays in the chromatogram, make sure that the following settings are defined for the fraction collector:

Calculate the delay time or dead volume as described in section To perform delay time or dead volume calibration above.
Specify the required fractionation mode.

Note:

It is not recommended to use the fractionation modes By_Time or By_Volume with the mode By_Peak in the same sample run.

Make sure that the dead time of the first detector (UV detector) and the offset times of the second detector (FL detector) and the third detector (FC_DelaySensor) are set correctly on the General tab page of the QNT editor. Otherwise, the overlay in the Chromeleon Chromatogram Plot will not be displayed properly.
Within one sequence, only one fraction tray can be used. If more trays are used, samples will not be displayed accurately on the Fraction Tray display.
Wait until the sample run has finished to view a complete and up-to-date visualization of the fractions in Chromeleon.

Display and Labeling of Fractions

After collecting fractions during an analysis, you can track in the chromatogram and the report which substances were collected in which tube. For details, refer to:

Tracking Fraction Collection in the Report

Tracking Fraction Collection in the Chromatogram

Supported trays and tray combinations

100 x 2 mL and 40 x 2 mL vials

100 x 2 mL and 15 x 6 mL vials

3 trays 40 x 2 mL vials

3 trays 15 x 6 mL vials

2 well plates + 10 vials

2 well plates + 10 vials + 40 x 2 mL vials

2 well plates + 10 vials + 15 x 6 mL vials

4 well plates standard tray

2 well plates + 10 funnels

2 well plates + 10 funnels + 40 x 2 mL vials

2 well plates + 10 funnels + 15 x 8 mL vials

214 x 12 mm tubes

126 x 16 mm tubes

60 x 25 mm tubes

40 x 30 mm tubes

2 well plates + 10 vials, supports well plates of 50 mm height

Further Information

For detailed information on fraction collection using the 1200 HPLC Fraction Collector, refer to the operation manual and the relevant application notes provided by Agilent.

For information on the additional commands and properties for fraction collection provided in Chromeleon, refer to the Commands dialog box (F8).