Page 262 - Vogel's TEXTBOOK OF QUANTITATIVE CHEMICAL ANALYSIS

8 COLUMN AN0 THIKLAVER LlilUlO CHROMATOCRAPHV

Chemicals. Indicator solutions ( - 0.1 per cent, uq.). Bromophenol blue; Congo
red; phenol red.
Mixture (M) of above three indicator solutions.
Developing solvent. n-Butanol-ethanol-0.2M ammonia (60:20:20 by volume).
chromatographic grade solvents should be used.
Procedure. Pour the developing solvent into the chromatographic tank to a
depth of about 0.5 cm and replace the lid. Take a prepared plate and carefully
'spot' 5 pL of each indicator on the origin line (see Section 8.6, under Sample
application) using a micropipette. Allow to dry, slide the plate into the tank
and develop the chromatogram by the ascending solvent for about 1 h. Remove
the plate, mark the solvent front and dry the plate in an oven at 60 OC for about
15 min. Evaluate the R, value for each of the indicators using the equation
Distance compound has moved from origin
R, =
Distance of solvent front from origin
Take a second prepared plate and 'spot' three separate 5 pL of mixture M
on to the origin line using a micropipette. Place the dry plate into the tank,
replace the lid and allow the chromatogram to run for about 1 h. Remove the
plate, mark the solvent front and dry the plate at 60 OC for about 15 min. Identify
the separated components on the basis of their R, values.
Carefully scrape the separated bromophenol blue 'spots' on to a sheet of
clean smooth-surfaced paper using a narrow spatula (this is easier if two grooves
are made down to the glass on either side of the spots). Pour the blue powder
into a small centrifuge tube, add 2 mL of ethanol, 5 drops of 0.880 ammonia
solution, and stir briskly until the dye is completely extracted. Centrifuge and
remove the supernatant blue solution from the residual white powder. Repeat
this procedure with the separated Congo red and phenol red 'spots'.
An alternative elution technique is to transfer the powder (e.g. for bromophenol
blue) to a glass column fitted with a glass-wool plug or glass sinter, and elute
the dye with ethanol containing a little ammonia. The eluted solution, made up
to a fixed volume in a small graduated flask, may be used for colorimetric/
spectrophotometric analysis of the recovered dye (see Chapter 17). A calibration
curve must, of course, be constructed for each of the individual compounds.

For References and Bibliography see Sections 9.9 and 9.10.

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