Aarre Kellomaki University of Turku Turku, Finland
I
A Simple Apparatus for Teaching the Principles of Chromatography
Although chromatography is extensively used in chemistry, it is difficultto find simple but satisfactory means for teaching its principles. The results obtained with paper chromatography or classical column chromatography are so inaccurate and unreproducible that they do not increase students' confidence in these techniques. For these reasons we have developed a modification of liquid column chromatography which has been successfully employed for teaching purposes in our laboratory for three years. The basic idea of this modification is to follow the elution of electrolytes by' measuring continuously the conductivity of the effluent. Apparatus and Procedure The apparatus is set up as shown in Figure 1. The vertical glass tube is an ordinary 50-ml buret with a, glass stopcock. The column is packed with Sephadev G-10 gel, which is much used for gel filtration in biochemistry'. The stock solutions of electrolytes are 0.2 M. In addition, a saturated aqueous solution of the macromolecular dye Blue Dextrrtn is needed. When the experiment is begun, the water level is lowered to the upper surface of the packing, and air bubbles are removed from the surfaces of the conductivity cell. The mixture to be examined is made by mixing 0.5-ml portions of two or three stock solutions of the electrolytes mentioned in the table and a few drops of the dye solution. The mixture is pipetted on the packing and is absorbed onto the gel by lowering the liquid level. The substances are eluted with ordinary tap water at a rate of I s 1 5 ml/hr and the solution flowing from the conductivity cell is collected in a graduated cylinder. Blue Dextran leaves the column first. It does not conduct electricity but is considered to have been eluted a t the moment when the intensity of the color between the plates of the cell is a maximum. The precision when judging the maximum intensity of the color in the autflowing liquid may be improved by collecting fractions of constant volume and comparing these with a series of standard dilutions. After eaoh conductivity maximum (peak), several drops of the emerging solution are taken for the identification of the salt with suitable reagents. The experiment is continued until all the salts have been eluted. The column is immediately ready for a new experiment without further treab ment.
V ,is best determined by filling the apparatus with water before adding the gel to the column and draining the water out and weighing it. V , is calculated from the weight and density of the dry gel, which is given by the manufacturer. The volume of effluent that has emerged from the column when the peak conductivity of a salt is recorded is called the elution volume V , of the salt in question. The void volume Vois equal to the elution volume of a substance like Blue Dextran which is completely excluded by the gel. The volume V t can now be calculated from the values of V,, Vo, and VOusing eqn. (1). I n order to make the experiment easier and faster for the students to carry out, the apparatus is not taken apart. Therefore the total volume of the column and the weight and density of the dry gel have to be given to the students. The elution volume of a substance is roughly constant when the same column is used. To compare results obtained with different columns, several parameters are calculated. First, there are two relative elution volumes: V,/Vo and V./V,. The inverse of V,/Vois called the retention constant. Second, the results can also be interpreted in terms of the fundamental equation of partition chromatography V.
=
+ K.V.
Vo
(2)
where K is the partition coefficient of the solute between the mobile and stationary phases and V sis the volume of the stationary phase. V , may be taken to be either the volume of the whole gel phase or the volume of
8
Water Tap
Treatment of the Results
The total volume V , of the chromatographic column is the sum of three volumes2. The gel matrix occupies the volume V,, and the water within the gel particles the volume V,. The third volume is the void volume Vo,which is the volume of the water between the gel grains and the volume of the water between the lower surface of the gel bed and the plates of the conductivity cell. We may thus write
DFIWHURST, F., J. CBEM.EDUC., 46,864 (1969). "Sephadex@--Gel Filtration in Theory and Practice," edited by Pharmacis Fine Chemicals, Uppsals, Sweden, 1966.
Figure 1.
Appwotur.
Volume 49, Number 2, February 1972
/
139
Typical Values of Chromatographic Parameters Computed from Experimental Data Collected by Students
Substance Blue Dextran EDTA (disodium salt) (COOK), NalSO, KsFe(CN)e KCl, NaCl KBr KI KSCN, NaSCN
V.
V
V
VJVt
1.3 1.35 1.4 1.65 1.7 2.0 2.6 3.2
0.44 0.46 0.48 0.56 0.58 0.68 0.88 1.10
K,,
Ks
015 0.18 0.21 0.33 0.36 0.52 0.82 1.15
0.23 0.27 0.32 0.50 0.55 0.77 1.23 1.73
17 ml (= Vo) 22 23 24 28 29 34 44 .55
The experiments were performed with a column of Sephadex G-10 (19 g dry gel) 50 ern high and 1 om in diameter. The volumes VO,Vi, VD,and VAwere 17, 22, 11, and 50 ml.
Figure 2.
Actudchromatogromr.
liquid in the gel particles. Accordingly, there are two partition coefficientsK., and Kd K.,
Vs - vo v, - vo
= -
and
Some Typical Student Results
In our experiments the Sephadex gel seems to act partly by functioning as a molecular sieve and partly
140
/
Journal of Chemical Education
by directly interacting with the solutes. The anion of an alkali metal salt largely determines the elution volume of the salt; the cation exerts only a slight influence. Salts with a common anion but different cations are not separated. Salts with large anions are eluted soon after Blue Dextran. Though our column is short, it partly separates alkali metal halides. This may be of value in analytical practice. The order of their elution, chloride, bromide, and iodide, is exceptional. Thiocyauate is eluted a t a low rate and its peak exhibits tailing. Experiments with salts of the alkaline earth metals, copper, and iron were not so successful as those with alkali metal salts (iron, for example, forms complexes with dextran). Some typical results are presented in the table. Actual chromatograms are reproduced in Figure 2. The distance from a point on the chromatogram to the base line is roughly proportional to the concentration and, accordingly, the area of a peak is roughly proportional to the amount of eluted substance. The dependence of the equivalent conductivities of electrolytes on concentration naturally complicates matters. Therefore, if the aim is to analyze quantitatively mixtures containing unknown amounts of salts, the apparatus must be calibrated with samples of known composition.
