Basic Information on SEC
Size Exclusion Chromatography (SEC) separates solutes in a sample according to molecular size of solutes. Pores of packing materials are used for the separation. When performing SEC, it is assumed that no interaction between solutes and packing material exists. Solutes to be analyzed by SEC mode have different aspects such as hydrophobic or hydrophillic, polar or non-polar and ionic or non-ionic, therefore, the selection of the eluent suited to the solutes is very important to realize pure SEC mode.
Features of SEC
1) No interaction between solutes and packing material is assumed.
2) To prevent the interaction, selection of appropriate eluent is essential.
3) The order of elution is the order of molecular size of solutes (from larger to smaller molecular size.)
4) The solutes whose molecular size is larger than the pore size of packing material cannot be separated each other. The upper limit of the molecular size which can be separeted is called as “exclusion limit”. The exclusion limit is peculiar to column type.
5) Elution volume which corresponds to exclusion limit is call as “void volume(Vo)”, which is a very important characteristic of SEC column.
6) The total volume of pores is called as “inner volume(Vi)”. The elution volumes of all solutes by pure SEC mode must exist between Vo and Vo + Vi.
Causes which prevent SEC mode
When performing SEC mode analysis, the most important thing is how to realize pure SEC mode. For the analyses of polystyrene and saccharides, the optimal conditions to realize pure SEC mode are well-known. However, for some samples, it is difficult to find the optimal condition. And, sometimes, chromatograms which do not act on SEC mode appear or only poor reproducibility can be obtained.
When pure SEC mode is performed, the calibration curve exist in the range of Vo and Vo + Vi as shown by the solid line in the right figure. However, when pure SEC mode is not performed, the calibration curve shown by the broken line can be obtained.
In case (A), some solute elute before Vo. The possible causes of this too fast elution are as follows:
* When the sample is ionic and the eluent is not an appropriate one, this phenomenon may happen. Please refer to SEC Analysis of Ionic Sample. Even when the elution volumes are in the range of Vo and Vo + Vi, this may be the cause of poor repuroducibility.
* When the aggregation of sample is taken place, this phenomenon may happen.
* When the ion-exclusion with packing material is taken place, this phenomenon may happen.
In case (B) and (C), solute must be adsorbed by the packing material. Such adsorption is mainly caused by hydrophobic or phdrophillic interaction and sometimes by hydrogen bond or ionic bond. The way t prevent such adsorption is as follows:
* To prevent hydrophobic or phdrophillic interaction, an eluent which has higher solubility to the solute and/or higher affinity to the packing material should be used.
* For some ionic polymers, addition of LiCl or LiBr increases the solubility and may solve the problem.
* When the sample is a polymer which has weak ionic group such as -COO–, the affinity of the polymer can be largely affected by the degree of dissociation. In such case, the degree of dissociation should be stabilized by using some buffer.
The solubility of solvents which are commonly used for SEC analysis is shown below:
| Solvent | Chemical formula |
Boiling
point (deg-C) |
Viscosity
cp, (25deg-C) |
Transmission
limit (nm) |
Refractive
index nD25 |
|---|---|---|---|---|---|
| Acetic acid | CH3COOH |
118
|
1.1
|
–
|
1.372
|
| Acetonitrile | CH3CN |
82
|
0.34
|
190
|
1.344
|
| Acetone | CH3COCH3 |
56
|
0.30
|
330
|
1.359
|
| Carbon tetrachloride | CCl4 |
77
|
0.90
|
265
|
1.466
|
| Chloroform | CHCl3 |
61
|
0.53
|
245
|
1.443
|
| Cyclohexane | C6H12 |
81
|
0.90
|
200
|
1.427
|
| Dichloroethane | CH2ClCH2Cl |
83
|
0.78
|
228
|
1.445
|
| Dichloromethane | CH2Cl2 |
40
|
0.41
|
233
|
1.424
|
| Dimethylformamide | (CH3)2NOCH |
153
|
0.80
|
268
|
1.428
|
| Dimethylsulfoxide | (CH3)2SO |
189
|
2.00
|
268
|
1.477
|
| Dioxane | C4H8O2 |
101
|
1.2
|
215
|
1.422
|
| Ethanol | C2H5OH |
78
|
1.08
|
210
|
1.361
|
| Ethyl acetate | CH3COOC2H5 |
77
|
0.43
|
256
|
1.370
|
| Ethylene glycol | HOCH2CH2OH |
182
|
16.5
|
–
|
1.431
|
| n-Hexane | CH3(CH2)4CH3 |
69
|
0.30
|
190
|
–
|
| Isopropyl alcohol | CH3CH(OH)CH3 |
82
|
1.9
|
205
|
1.384
|
| Isopropyl ether | (CH3)2CHOCH(CH3)2 |
68
|
0.38
|
220
|
1.365
|
| Methanol | CH3OH |
65
|
0.54
|
205
|
1.329
|
| Methyl ethyl ketone | CH3COC2H5 |
80
|
0.38
|
329
|
1.381
|
| n-Propanol | CH3CH2CH2OH |
97
|
1.9
|
240
|
1.385
|
| THF | C4H8O |
66
|
0.46
|
212
|
1.408
|
| Toluene | C6H5CH3 |
110
|
0.55
|
285
|
1.496
|
| Water | H2O |
100
|
0.89
|
–
|
1.333
|
| 1,2,4-Trichlorobenzene | C6H3Cl3 |
213
|
1.89
|
–
|
1.5717
|
| o-Dichlorobenzene | C6H4Cl2 |
180
|
1.26
|
–
|
1.5515
|
| Hexafluoroisopropanol | (CF3)2CHOH |
58
|
1.62
|
–
|
1.275
|
| Solvent | chemical formula | delta | P’ |
|---|---|---|---|
| Cyclohexane | C6H12 | 8.2 | -0.2 |
| n-Hexane | CH3(CH)3 | 7.3 | 0.1 |
| Carbon tetrachloride | CCl4 | 8.6 | 1.6 |
| Isopropyl ether | (CH3)2CHOCH(CH3)2 | 7.0 | 2.4 |
| Toluene | C6H5CH3 | 8.9 | 2.4 |
| Dichloromethane | CH2Cl2 | 9.6 | 3.1 |
| Dichloroethane | CH2ClCH2Cl | 9.7 | 3.5 |
| Isopropyl alcohol | CH3CH(OH)CH3 | 10.2 | 3.9 |
| THF | C4H8O | 9.1 | 4.0 |
| n-Propanol | CH3CH2CH2OH | – | 4.0 |
| Chloroform | CHCl3 | 9.3 | 4.1 |
| Ethanol | C2H5OH | 11.2 | 4.3 |
| Ethyl acetate | CH3COOC2H5 | 8.6 | 4.4 |
| Methyl ethyl ketone | CH3COC2H5 | – | 4.7 |
| Dioxane | C4H8O2 | 9.8 | 4.8 |
| Acetone | CH3COCH3 | 9.4 | 5.1 |
| Methanol | CH3OH | 12.9 | 5.1 |
| Acetonitrile | CH3CN | 11.8 | 5.8 |
| Acetic acid | CH3COOH | 12.4 | 6.0 |
| Dimethylformamide | (CH3)2NOCH | 11.5 | 6.4 |
| Ethylene glycol | HOCH2CH2OH | 14.7 | 6.9 |
| Dimethylsulfoxide | (CH3)2SO | 12.8 | 7.2 |
| Water | H2O | 21.0 | 10.2 |
delta : Solubility parameter
P’ : Rohrschneider’s polarity parameter
Please refer to the following tables for the classification of solvents in terms of capability of being replaced with original in-column solvent. However, frequent replacement of in-column solvent may shorten column life, and is, therefore, not recommended. We can replace the in-column solvent with another solvent such as o-dichlorobenzene, ethyl acetate, dimethylacetamide or methylpyrrolidon upon your request.
(Standard Organic SEC (GPC) columns)
(Downsized GPC columns and Semi-micro GPC columns)
(Linear Type : LF series)
Basically, the sample should be dissolved in the same solvent that is to be used as the eluent.
1) Dissolving the sample in such solvent will make the blank peaks as small as possible.
2) In case the sample has a molecular weight of 1,000,000 minimum, soak it in such solvent for 12 to 24 hours to let it swell. After the swelling, stir the solvent gently for dissolution.
Strong agitation or use of an ultrasonic bath for dissolution will degrade such sample.
3) In case the sample is a polymer, its concentration in the solution and the injection volume should be 0.05 to 0.5% and 50 to 100micro-L, respectively.
If the concentration is higher, the retention volume of the sample will increase. The optimum concentration changes with the molecular weight and the viscosity. The following table gives the molecular weight vs. the optimum concentration.
| Molecular weight | Sample concentration (W/V%) |
Injection volume per column | |
|---|---|---|---|
| KF-600 series | KF-800 series | ||
|
< 5,000
|
less than 1.0
|
micro-L | to 100 micro-L |
|
5,000 to 25,000
|
less than 0.5
|
||
|
25,000 to 200,000
|
less than 0.25
|
30 micro-L | |
|
200,000 to 2,000,000
|
less than 0.1
|
||
|
> 2,000,000
|
less than 0.05
|
||
4) In case the sample is organic, it is desirable that the concentration be 1% maximum and the injection volume, 50micro-L maximum.