Question

Case C Thin Layer Chromatography 1. TOXI-LAB is a screening method that uses: chromatography (column / planar) The first step consists of the extraction of drug metabolites from biological fluids when we place the sample in a test tube containing a mixture of solvents and buffering salts that cause the extraction of basic and neutral drugs. The solvent extract of the sample is concentrated by heat and evaporation, then is deposited onto a small disc of chromatographic media (small circle of fier pape). This dried disk is inserted in an opening of a Toxi Gram paper (small opening at the bottom of the paper is the area to place the disk with patients sample). The Toxi gram paper has lanes that already contain standards of different drugs which show spots with a specific Rf related to the presence of those substances. Spots are revealed when the Toxi gram paper after the chromatography is exposed to developing reagents. 2. In this chromatography the organic solvent in which the Toxi Gram paper is immersed is considered to be the the (stationary/mobile) phase, and the Toxi Gram paper is phase In the process drug metabolites present in the disk that contains the sample elute from the disk and migrate reaching a specific position on the chromatogram. The standards give us the reference positions for the common drugs included in the standard lanes. After the spot detection process when the chromatogram is dipped into chromogenic reagents the identification process is based on matchinga drug spot in the unknown zone with an adjacent standard drug spot. For the match the spot should have the same Rf, size, shape and color characteristics of the standard through all the stages of the developing process 3. Indicate how the Rf is calculated: In a Toxi Lab, the following standards had these Rf values Lane #1-Acetaminophen 0.20 Lane #2-Diazepam 0.6 4. Lane # 5-Amphetamine 0.43 Lane #6-imipramine 0.85 The patients sample were in Lane #3 and Lane #4; if the solvent front traveled 8 cm and the spot in the unknown patient sample in lane #3 traveled 1.6 cm while the spot in the unknown disk on Lane #4 traveled 6.8 cm. Calculate the Rf for the spots detected on lane #3 Rf- Indicate the preliminary report for samples in those lanes Lane #3 Report 5. and Lane #4 Rf . Lane #4 Report: 6. The TLC screnning procedure provides a 7. List two advantages of the TLC technique for testing drugs of abuse 8. Use the information of standards and unknowns to draw a diagram of that TLC chromatogram (qualitative/quantitative) report

Answer 1

1. planar chromatography

it is  hypothesized that planar chromatography is effective than column chromatography, because planar chromatography can effectively work as 2D(Two-Dimensional) method, which can separate many analytes that are components in bilogical samples like blood, urine etc.

2. Solvent is the mobile phase and Toxi gram paper is the stationary phase

Stationary phase: a solid support. different stationary phase are used based on chromatographic techniques applied. For paper chromatography: stationary phase is uniform absorbent paper, In TLC Silica gel coated TLC plates acts as stationary phase

Mobile phase: Liquid or Gas acts as mobile phase. Different solvents from polar(Acetonitrile) to non polar solvents (Hexane), In Gas chromatography gases like argon or helium acts as mobile phase.

3. Rf value: It is the retardation factor/ retention value in chromatography, it is the ratio of distance travelled by solute(sample under testing or standard compounds) to distance travelled by the solvent that is mobile phase also called as solvent front

In the above chromatogram the distance travelled by solute is calculated using Rf formula.

Rf= distance travelled by solute/Distance travelled by solvent

Distance travelled by solvents = distance travelled by solvent* Rf

Rf= 0.20,0.6, 0.43.0.85

distance travelled by solvent = 8

So distance travelled by solute are 1.6 cm, 4.8 cm, 3.44 cm and 6.8 cm respectively