Determination of mobile phase for an unknown substance

By - 03/24/2005
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Dr.S.N.Meyyanathan

Principle:


The factors involved in the determination of mobile phase are nature of substance, nature of stationary phase and mode of chromatography i.e. normal phase or reverse phase.

Single solvent or mixture of solvents can be used as mobile phase. Mobile phase composition can be selected by trial and error method only but with the review of literature and solubility of unknown substance, mobile phase composition can also be determined.


Lists of solvents with elution power values are as follows:




























































SOLVENTS ELUTION POWER
1) Hexane 0.0
2) Carbon tetra chloride 1.6
3) Toluene 2.4
4) Xylene 2.5
5) Benzene 2.7
6) Tetra hydro furan 3.5
7) Chloroform 3.9
8) Iso propyl alcohol 4.0
9) Ethyl acetate 4.4
10) 1,4 - Dioxan 4.8
11) Acetone 5.1
12) Methanol 5.1
13) Ethanol 5.2
14) Acetonitrile 5.8
15) Acetic acid 6.2
16) Dimethyl formamide (DMF) 6.4
17) Dimethyl sulfoxide (DMSO) 7.2
18) Water 9.0



 Procedure:



  • Prepare slurry of 150 gram of silica gel GF in 300 ml of distilled water and pour the slurry uniformly on glass TLC plates and after setting the plates are then dried in an oven at 120-degree C for 20 minutes or use pre coated TLC plates.

  • Check the solubility of unknown substance in different solvents as mentioned in the table.

  • Solvents in which the substance was soluble are added into different beakers individually lined with filter paper, covered with watch glass and keep it for 30 minutes for saturation of beaker so as to get the clear spots in short time with better resolution.

  • Mark a base line 2-cm above the surface of dried TLC plate with pencil and spot the solution of substance from different solvents using capillary tube on the base line.

  • The plate is then kept inside the beaker such that lower end of the plate dips into the solvents in the respective beaker and spots of substance should not touch the solvents.

  • The solvent now travels along the stationary phase i.e. TLC plate by capillary action and when it reaches three fourth of the length of plate, remove the plate from the beaker, mark the solvent front with pencil and dry it.

  • The plate is then observed under UV light at 254 nm or 366 nm or in iodine chamber.

  • If the spot is on or near the solvent front add non-polar solvents which should be miscible with the selected solvent and if the spot is on or near the base line add polar solvents which should be miscible with the selected solvent.

  • The spot of the substance is then marked or rounded with pencil and Rf value is calculated.


Rf = distance travelled by the substance / distance travelled by the solvent front


Ideal Rf values for any substance should be between 0.2 and 0.8.


{mospagebreak title=About the author}


Author Contact Information:




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Dr S.N.Meyyanathan



Professor,

Dept.of Pharmaceutical Analysis,

J.S.S.College of Pharmacy,

P.B.20, Rocklands, Ootacamund 643 001, Tamilnadu, India.

Phone : 0423-2443393 (O)

Fax : 0423-2442937

E-mail: meyys@rediffmail.com