1. PERISTALTIC PUMP TYPE

This contribution describes the up-grading of the spectrometer SpektraTM into simplified liquid chromatograph for educational purposes. The chromatograph was tested for the separation of dyes in natural Mint aroma. 

Apparatus and equipment

  • spectrometer SpektraTM,
  • a peristaltic pump with tubes for 0.25 ml/min flow rate. We used ISMATEC MS-CA2840 pump  with constant number of turns, a Tygon tubing  with internal diameter of Φi 0.25 mm.
  • a transparent polymer tubing with 0.8 -1.0 mm internal diameter for making a flow cell for the spectrometer and for joining the parts of the chromatograph,
  • a 2 ml Pasteur pipette PLASTIBRAND with the top of the bulb cut off and a tip cut off so that  only  approx. 0.5 cm  of narrowed tubing is left
  • a laboratory stand, clamps,
  • 50 ml measuring flask,
  • 100 ml volumetric flask,
  • 25 ml beaker,
  • a larger syringe,
  • a tubing to connect  the tip of the syringe with the lower end of the column,
  • a micropipette for dispensing 20 ml volumes, or, in a simplified version a dropping bottle  which allows for dispensing drops of comparable volumes,
  • a dropper,
  • a stopwatch,
  • (optional: a computer with interface for automatic data acquisition, or an integrator).
     

Reagents

  • ammonia (25 %),
  • butane-1-ol (ρ = 0.808 kg/l),
  • propane-1-ol (ρ = 0.8 kg/l),
  • Silica-gel (Fluka, Kieselgel 60, 230-400 mesh, particle size from 0.04 mm to 0.063 mm),
  • sand,
  • glass wool,
  • Mint, natural aroma (Tovarna arom in eteričnih olj, d.d.).
     

Hazards
 

Ammonia causes burns. Dangerous for aquatic organisms. In contact with skin or eyes irrigate with plenty of water and seek medical help. Wear protective glasses and gloves.   Do not dispose of the liquid into the environment.
Risk phrases: 34-50, Safety phrases: 26-36/37/39-45-61
 

Butane-1-ol is a flammable and harmful substance if ingested. It is a skin and respiratory system irritant and harmful in contact with eyes. The fumes can cause dizziness and confusion. Keep the chemical in air-tight containers and well ventilated places. If in contact with eyes, irrigate with plenty of water and seek medical help. If ingested, seek medical help and show safety signs on the label. Wear protective glasses and gloves. Do not dispose of the liquid into the environment.  
Risk phrases: 10-22-37/38-41-67, Safety phrases: 7/9-13-26-37/39-46

Propane-1-ol is flammable and irritant. If inhaled it causes ataxia, confusion, nausea, headache, inebriation. It causes dryness of skin and skin redness and blurred vision. Use protective glasses and gloves.  
Risk phrases: 11-41-67, Safety phrases: (2-)7-16-24-26-39

 

Preparing the mobile phase

Prepare the mobile phase in a 100 ml flask according to the volumes  given in the table below.   

Table 1. Volumes for preparing the mobile phase

deionized water

propane-1-ol

ammonia (25 %)

butane-1-ol 

15 ml 

45 ml

7.5 ml

25 ml 

Stir the solution well and keep it in a sealed flask.


Assembling the liquid chromatograph

Use a transparent polymer tubing as a flow cell, which will be inserted into the measuring site in the SpektraTM spectrometer. The diagram for making a flow cell is shown in Picture 1. Insert the flow cell into the measuring site and fix it, making sure that it stays in place during the experiment.  

Picture 1. Scheme of the flow cell

Use the Pasteur pipette (it is described in the »Apparatus« section above) as a column. Insert a plug made of glass wool into the lower part of the column (do not touch glass wool by hands, use tweezers!).

Mix in a beaker 5 g of  Silica-gel and 10 ml of the mobile phase. Fill the column with the prepared mixture of Silica-gel and mobile phase. Compact the Silica-gel layer with the help of the syringe which is connected with the bottom part of the column causing underpressure. Make sure that the level of the mobile phase does not fall below the level of Silica-gel.  The Silica-gel in the column prepared according to the described procedure, should not be more than  2.5 cm high. Cover the Silica-gel layer with a 0.5 cm layer of finely ground sand. Fix the column on the stand and attach it to the inlet tubing of the flow cell. Connect the outlet of the flow cell to the   peristaltic pump tube. Place the other end of the tube of the peristaltic pump into the beaker which serves as a receiver for discard solution.  

Protect the measuring chamber with appropriate cover (i.e. aluminium foil) in order to prevent the effect of the surrounding light on the measuring values of the spectrometer. See the picture of the assembled chromatograph on Picture 2. Optionally you can connect the analogue output of the spectrometer via a suitable interface with a computer, or use an integrator. Measurements can be taken manually as well.

Picture 2. Liquid chromatograph


Using the chromatograph for separating the dyes of natural Mint aroma

Natural Mint aroma contains  E102 and E131 dyes. Search the Internet to find the information about these dyes.  After you have assembled the chromatograph and checked that the flow of the mobile phase is 0.25 ml/min, you can start with separation of the dyes.  

The column must be filled with the mobile phase. Switch on the spectrometer and select blue or red LED. Set the intensity of the light emitter to its maximum.  Switch on the pump and propel through the system the mobile phase until its volume equals to the triple volume of the stationary phase.    

Set the  transmittance to 100.0. When the level of the mobile phase has reached the level of the sand dispense 20 ml of natural Mint aroma onto the top of the sand layer and start the stopwatch. Use a dropper, carefully adding the mobile phase along the wall of the column, taking care that the level of the liquid does not fall under the level of Silica-gel. Record the results in 10 seconds intervals.   

The separation of components in the column is shown in Picture 3.

 

Picture 3. Separating dyes in the column   

Convert the transmittance into absorbance and draw a graph. Since the components are not equally coloured you need to repeat the experiment, using another LED (if you used blue LED first, take red LED next).  

Picture  4 shows two examples of chromatograms obtained by computerized data acquisition and measurement.

Picture 4. Chromatograms which were obtained by  using blue and red  LED.
 

Developed and prepared by: Nataša Gros and Domen Klančar, University of Ljubljana, Faculty of Chemistry and Chemical technology