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How to Calculate Dead Time on the LC Column

Chromatography is widely used in the chemical laboratory to determine the identity of an unknown compound. The unknown compound flows through the packing material of the column and interacts with the particles. The interactions between the particles and the compounds in the sample are based on their attraction or solubility to one another. Molecules that have no interaction to the particles of packing material are allowed to pass through the packing material unhindered. The time required for molecules to pass from the injection point into the system to the maximum peak of an unreactive molecule is the dead time of the column.

Things You'll Need

Unreactive molecule
LC chromatography instrument
Sample syringe

Instructions

- 1
  Spike the unknown sample with a small amount of a compound that is not retained by the column material. The compound will pass through the column without interacting with the packing material in the column.
- 2
  Load the syringe with approximately 20 microliters of sample and insert the syringe needle into the injection port. Load the sample loop of the valve by emptying the syringe into the valve.
- 3
  Turn the chromatography instrument on and start the mobile phase. The mobile phase flows through the instrument and carries the sample through the column and detector ending in the waste container.
- 4
  Activate the valve, which allows the sample to enter the flowing path of the solvent. The solvent carries the compounds in the sample onto the top of the column and separation begins.
- 5
  Monitor the signal output of the detector and identify the first peak in the chromatograph. The difference between the injection time and the maximum of the first peak, represented by the spiked compound, is the dead time.
- 6
  Measure the distance between the injection point and the maximum height of the first peak. Multiply the distance by the speed of the paper or axis to find the dead time.

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