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Fraction ScheduleFree in-browser calculator

Microdialysis Fraction Planner.

Enter flow rate, collection interval, and probe specs. Get a complete fraction schedule with dead volume delay, evaporation risk assessment, and printable bench sheet.

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Validated2026-04-05
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Flow Parameters

Vial & Probe

Dead Volume (Outlet Tubing)

Computed Dead Volume3.39 µL

Summary

Volume per Fraction

15.00µL

Vial Fill

5.0%

Dead Volume Delay

3.39 min

Total Volume

180.00 µL

Total Duration

180 min
Vial Utilization5.0% of 300 µL

Probe recovery: 18% estimated recovery through 2 mm membrane (20 kDa MWCO). Dialysate concentration ≈ 18% of tissue extracellular concentration.

Collection Timeline

12 fractions over 180 min(hatched = 3.39 min dead volume delay)
0 min183 min

Collection Schedule

FractionStart (min)End (min)Volume (µL)Cumulative (µL)
#101515.0015.00
#2153015.0030.00
#3304515.0045.00
#4456015.0060.00
#5607515.0075.00
#6759015.0090.00
#79010515.00105.00
#810512015.00120.00
#912013515.00135.00
#1013515015.00150.00
#1115016515.00165.00
#1216518015.00180.00

Note: First 3.39 min of collection reflects dead volume transit (tubing 0.12 mm ID × 30 cm).

When to use

  • Calculate fraction volumes from flow rate and collection interval to verify compatibility with your analytical method's minimum sample requirement
  • Generate a complete collection schedule with fraction numbers, start/stop times, and cumulative volumes for programming your fraction collector
  • Estimate dead volume and time lag from outlet tubing dimensions to correctly align fraction timestamps with experimental events
  • Check whether your planned fraction volume is at risk of significant evaporative loss based on collection temperature and vial type
  • Compare protocols at different flow rates to find the optimal trade-off between temporal resolution and relative recovery for your analyte
  • Plan mixed-interval collection schedules with shorter fractions during experimental events and longer fractions during baseline and recovery periods

Do not use for

  • Quantitative prediction of relative recovery — recovery depends on in vivo tissue properties, probe-tissue interface maturation, and analyte-specific membrane interactions that require empirical calibration (no-net-flux or retrodialysis methods)
  • Online or continuous-flow analytical setups where dialysate flows directly to a detector (e.g., biosensor or segmented-flow systems) without discrete fraction collection
  • Push-pull perfusion or open-flow microperfusion systems that do not use a semi-permeable membrane and have fundamentally different fluid dynamics

Account for dead volume delay before aligning data with events

The time lag from dead volume can exceed your fraction interval, causing the sample in a given vial to actually represent an earlier time window. Always calculate dead volume delay (= tubing volume / flow rate) and subtract it from collection timestamps. Failure to correct for dead volume is one of the most common sources of temporal misalignment in published microdialysis data.

Verify fraction volume exceeds analytical minimum plus pipetting margin

If your HPLC requires a 10-microliter injection, a 10-microliter fraction leaves zero margin for pipetting error, duplicate injections, or evaporative loss. Plan for at least 50% excess volume — a 15-microliter fraction for a 10-microliter injection — or adjust your flow rate and interval accordingly. Running out of sample after a multi-hour in vivo experiment is irrecoverable.

Refrigerate fractions to prevent evaporation and analyte degradation

Microdialysis fractions are typically 2-40 microliters. At room temperature in open vials, evaporation can reduce volume by 10-30% over a 3-hour collection period, artificially concentrating analytes and introducing systematic bias. Use a refrigerated fraction collector (4-6 degrees Celsius) and consider adding antioxidant (e.g., perchloric acid for monoamines, EDTA for peptides) to the collection vials to prevent both evaporation and degradation.

Check for air bubbles before and during collection

Air bubbles in the inlet or outlet tubing disrupt flow continuity, causing irregular fraction volumes, loss of membrane perfusion, and gaps in the neurochemical time series. Degas your perfusion fluid, prime the system thoroughly before connecting the probe, and visually inspect the outlet tubing periodically during collection. A single bubble can invalidate an entire fraction, and multiple bubbles can compromise the whole experiment.

Use stabilization period before collecting experimental fractions

After probe insertion, tissue damage causes an acute neuroinflammatory response and artifactual neurotransmitter release that can persist for 30 minutes to several hours. Standard practice is to allow a 60-120 minute stabilization period after probe insertion (or overnight for chronic implants) before beginning baseline fraction collection. Fractions collected during the stabilization period should be discarded or analyzed separately, not included in experimental baselines.

1

Method

Fraction volume is computed as flow rate multiplied by collection interval. Dead volume is calculated from outlet tubing geometry using the cylindrical volume formula (pi * r^2 * L), and the resulting time lag is dead volume divided by flow rate. Evaporation risk is flagged when fraction volumes fall below empirically established thresholds for the selected collection temperature (room temperature: fractions below 3 microliters flagged as high risk; refrigerated at 4 degrees Celsius: fractions below 1.5 microliters flagged). The collection schedule generator produces a time-indexed table of fraction numbers, start and end times, individual and cumulative volumes, and annotates each fraction with its dead-volume-corrected event time. All computation is performed client-side — no data leaves your browser.

2

Validated

Last validated 2026-04-05. Calculations are designed for planning and documentation support; verify procurement decisions against manufacturer specifications or institutional SOPs.

3

How to cite

How to Cite

ConductScience Microdialysis Fraction Planner (v1.0). ConductScience, Inc. 2026. Available at: https://conductscience.com/tools/microdialysis-fraction-planner

This tool performs arithmetic calculations for fraction volume planning and dead volume estimation based on user-provided tubing dimensions and flow rates. It does not predict in vivo relative recovery, analyte stability, or tissue-specific diffusion properties. Collection schedules should be validated against your specific probe, analytical method, and experimental requirements before use in animal studies.

Microdialysis Sampling Principles

Microdialysis is founded on the principle of passive diffusion across a semi-permeable membrane implanted in living tissue. A perfusion fluid, chosen to match the ionic composition of the surrounding extracellular fluid, is continuously pumped through the probe lumen at a controlled flow rate, typically between 0.1 and 5.0 microliters per minute. Small molecules in the extracellular space diffuse across the membrane into the perfusate along their concentration gradient, while the continuous flow carries these molecules to an outlet tube where they are collected as discrete fractions. The technique was pioneered by Urban Ungerstedt in the 1970s and has since become the gold standard for in vivo neurochemical monitoring in freely moving animals. The key analytical parameter is relative recovery — the ratio of dialysate concentration to true extracellular concentration. Relative recovery is governed by the Bungay mass transport model, which accounts for resistances at the membrane, the extracellular space, and the probe-tissue interface. Recovery depends on flow rate (inversely), membrane area (directly), membrane material and MWCO, analyte diffusion coefficient, and tissue tortuosity. At typical experimental flow rates of 0.5-2.0 microliters per minute, relative recovery for small neurotransmitters ranges from 5-25%, meaning the dialysate represents a diluted but proportional sample of the extracellular milieu. This dilution factor must be considered when interpreting absolute concentrations, although most microdialysis studies report relative changes from baseline, making exact recovery less critical. The no-net-flux and retrodialysis calibration methods can be used to estimate in vivo recovery when absolute quantification is required.

Optimizing Flow Rate and Fraction Volume

The choice of flow rate and fraction collection interval represents the central trade-off in microdialysis experimental design: temporal resolution versus analytical sensitivity. Higher flow rates produce larger fraction volumes in a given time interval, providing more sample for analytical detection, but reduce relative recovery because the perfusate equilibrates less with the tissue. Lower flow rates improve recovery but yield smaller volumes that may fall below the analytical method's limit of detection or minimum injection volume. The fraction volume is simply the product of flow rate and collection interval: for example, 1.0 microliter per minute collected for 15 minutes yields a 15-microliter fraction. The minimum usable fraction volume is determined by the analytical platform — HPLC-ECD systems typically require 5-20 microliters for injection, while LC-MS/MS methods can work with as little as 1-5 microliters. When designing a collection schedule, researchers must also consider the number of fractions needed: a 3-hour experiment with 10-minute fractions generates 18 fractions per probe, while 5-minute fractions double that to 36 fractions, increasing analytical workload and consumables cost. For pharmacokinetic studies where drug concentrations change slowly over hours, 15-20 minute fractions are adequate. For fast neurotransmitter dynamics such as phasic dopamine release during operant behavior, shorter fractions (1-5 minutes) or even online detection systems may be necessary. A practical approach is to use longer baseline fractions (15-20 minutes) before the experimental manipulation, then switch to shorter fractions (5-10 minutes) during the period of interest, and return to longer fractions during the recovery phase. This mixed-interval protocol maximizes temporal resolution when it matters most while keeping total fraction count manageable.

Dead Volume and Temporal Resolution

Dead volume — the internal volume of the outlet tubing between the probe tip and the fraction collector — introduces a fixed time delay between the moment an analyte crosses the membrane and when it appears in the collected fraction. This delay, calculated as dead volume divided by flow rate, must be subtracted from fraction timestamps to accurately align neurochemical data with experimental events. For a typical setup with 30 cm of polyethylene PE-10 tubing (0.28 mm ID), the dead volume is approximately 18.5 microliters, creating an 18.5-minute lag at 1.0 microliter per minute — a delay that can be larger than the fraction interval itself, causing collected samples to reflect events from the previous collection period. Reducing dead volume requires using shorter outlet tubing, narrower-bore tubing (e.g., fused silica with 50-75 micrometer ID), or both. However, narrower tubing dramatically increases backpressure (fourth-power relationship with diameter), which can exceed the syringe pump's pressure limit or cause probe membrane failure. The optimal outlet tubing configuration balances dead volume against practical backpressure constraints. Fused silica capillary (75 micrometer ID) at 20 cm length gives approximately 0.88 microliters dead volume and a lag of only 53 seconds at 1.0 microliter per minute, but generates substantially more backpressure than standard PE tubing. Dead volume also affects temporal smearing: analyte pulses are dispersed by Taylor dispersion as they travel through the tubing, broadening sharp concentration transients into smoother peaks. This dispersion is minimized in narrow-bore tubing at higher flow rates. When planning an experiment, always calculate the dead volume delay and verify that it does not exceed your desired temporal resolution. If the delay is unacceptable, shorten the tubing, reduce the ID, or increase the flow rate (accepting the recovery trade-off).

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