Page 59 - Subyek Encyclopedia - Encyclopedia of Separation Science

54 I / CHROMATOGRAPHY/ Derivatization

Table 3 Factors affecting resolution in column chromatorgraphy

Value of N needed for Value of N needed for R S "1at
R S "1at k"3 for different different k values for "1.05 and
values of 1.10

N k "1.05 "1.10
1.005 1 150 000 0.1 853 780 234 260
1.01 290 000 0.2 254 020 69 700
1.02 74 000 0.5 63 500 17 420
1.05 12 500 1.0 28 220 7 740
1.10 3 400 2.0 15 880 4 360
1.20 1 020 5.0 10 160 2 790
1.50 260 10.0 8 540 2 340
2.00 110 20.0 7 780 2 130

illustrated by the data in Table 3, which indicate the
number of theoretical plates required for a separ-
ation. These data can be compared to the data in
Table 1, which indicates the number of theoretical
plates available for different chromatographic
systems. This is a clear indication of the need for
selectivity optimization in LC and SFC, and the more
Figure 16 Separation of aromatic compounds by MEKC using
relaxed constraints for GC. a 65 cm (effective length 50 cm) 50 m i.d. fused silica capillary
Resolution will initially increase rapidly with reten- and a mobile phase containing 30 mmol L sodium dodecyl
1
1
1
tion, starting at k"0, as shown in Figure 15. By the sulfate and 50 mmol L sodium phosphate/100 mmol L so-
time k reaches a value around 5, further increases in dium borate buffer (pH"7) at a field strength of 15 kV. (Repro-
retention result in only small changes in resolution. duced with permission from Terabe S (1989) Trends in Analytical
Chemistry 8: 129, copyright ^ Elsevier Science B.V.)
The optimum resolution range for most separations
occurs for k between 2 and 10. Higher values of
k result in long separation times with little concomi- totally retained by the micelles, t MC ; see Figure 16).
tant improvement in resolution, but they may be The intrinsic efRciency of MEKC is much higher
necessary to provide sufRcient separation space than column liquid chromatography, and optimiza-
to contain all the peaks in the chromatogram. tion of the separation factor depends on a differ-
The separation time is given by: ent set of parameters (changing surfactant type,
use of additives, etc). Large values of the retention
3
2
2
2
t R "(H/u) 16R S [ /( !1) ] (k 2 #1) /k 2 ) [5] factor are unfavourable for obtaining high resolution
since the additional term added to the resolution
equation tends to zero at high k values. The optimum
If the separation time (t R ) is to be minimized, then the
value of k for maximum resolution is around 0.8}5,
acceptable resolution should not be set too high 1/2
corresponding to (t M /t MC ) . The retention factor is
(R S "1); the separation factor should be maximized usually optimized by changing the surfactant concen-
for the most difRcult pair to separate; the reten- tration.
tion factor should be minimized (k"1}5) for the
most difRcult pair to separate; and the column Planar Chromatography
should be operated at the minimum value for the
plate height corresponding to the optimum mobile- For a single development under capillary-controlled
phase velocity. Sow conditions the TLC analogue of the general
resolution equation for column chromatography can
Micellar Electrokinetic Chromatography be expressed in approximate form as:
The resolution equation for MEKC is identical to R S "[(N 1 R F2 ) /4] [(k 1 /k 2 )!1)] (1!R F2 ) [6]
1/2
eqns [3] and [4] but contains an additional term,
(t M /t MC )/[1#(t M /t MC )k 1 ], to account for the limited where N 1 is the maximum number of theoretical
elution range (all solutes must elute between the re- plates available corresponding to the solvent
tention time of an unretained solute, t M , and a solute front position. The use of N 1 R F2 is only a rough