The standard elution procedure always begins with a “nonpolar” fraction (fraction 1) typically consisting of a single pure solvent, such as hexane (H) or cyclohexane. Successive fractions (fraction 2, 3, …) are constructed so that each one is slightly more polar than the one that came before. This is accomplished by mixing the nonpolar solvent with increasing amounts of a slightly more polar solvent, such as ethyl acetate (EA), ether, or dichloromethane.

The following table shows a typical set of 24 solvent fractions. The first one is pure hexane (0 EA: 100 H). After that fraction polarity steadily increases in small steps until the final one (40 EA: 60 H). If you look carefully, you will see that the initial polarity changes are smaller (1-2%) than the final changes (3-4%).

Fraction # | Nominal Composition (EA:H) |
Starting EA:H | EA to add (ml) |
---|---|---|---|

1 | 0:100 | 0:100 | |

2 | 1:99 | 1 | |

3 | 2:98 | 0.5 | |

4 | 4:96 | 0.5 | |

5 | 5:95 | 5:95 | |

6 | 6:94 | 1 | |

7 | 7:93 | 0.5 | |

8 | 9:91 | 0.5 | |

9 | 10:90 | 10:90 | |

10 | 11:89 | 1 | |

11 | 12:88 | 0.5 | |

12 | 14:86 | 0.5 | |

13 | 15:85 | 15:85 | |

14 | 16:84 | 1 | |

15 | 17:83 | 0.5 | |

16 | 18:82 | 0.5 | |

17 | 20:80 | 20:80 | |

18 | 22:78 | 2 | |

19 | 24:76 | 1 | |

20 | 26:74 | 1 | |

21 | 30:70 | 30:70 | |

22 | 33:67 | 3 | |

23 | 36:64 | 3 | |

24 | 40:60 | 2 |

If you suppose that it might be very time-consuming to make up 24 slightly different fractions from scratch, you would be right. A typical fraction size is 25 mL. To make fraction #20 would require mixing EA and H in such a way that we end up with 25 mL of a mixture that is precisely 26% EA and 74% H. Ouch.

Fortunately, this kind of precision is not required. The solvent ratios in the table are guidelines, and they do not need to be met exactly as stated. The more important thing is to obey the principle of *steady, but small, increases* in *polarity* from one fraction to the next.

The following instructions show you how to quickly make 4 consecutive fractions using just three pieces of glassware: a 100 mL graduated cylinder, a 25 mL graduated cylinder, and a disposable pipet that delivers roughly 1 mL of solvent per pipet load.

For our example, we will take as our goal making fractions #9-12. The polarity (composition) of these mixtures ranges from 10 EA: 90 H (fraction #9) to 14 EA: 86 H (fraction #12). Naturally, we begin with fraction #9 by making 100 mL of the 10:90 mixture that is needed (see fraction #9 entry in the **Starting EA:H** column) in the 100 mL graduated cylinder.

- Pour 90 mL of H into a 100 mL graduated cylinder. Add ~10 mL of EA so that the total volume is 100 mL. Mix the solution well. You can do this easily by pouring back-and-forth between the graduated cylinder and a 200 mL Erlenmeyer flask.

The next step is to use 25 mL of this 10:90 mixture to collect fraction #9. You don’t have to be precise.

- (Option A) Pour the 10:90 mixture in the graduated cylinder directly on to your column. Collect roughly 25 mL of liquid.
- (Option B). Pour 25 mL of the 10:90 mixture into the 25 mL graduated cylinder, then pour this aliquot on to the column. The volume you collect is fraction #9.

At this point, you should have ~75 mL of 10:90 solvent remaining in your 100 mL graduated cylinder. You need to add a small amount of EA to increase its polarity to the 11:89 mixture needed for fraction #10. According to the right-most column in the table (**EA to add**), you should add 1 mL of EA to the 75 mL.

- Add 1 pipet load of EA to the mixture in the 100 mL graduated cylinder. Mix well.
- Transfer ~25 mL of this mixture to your column to collect fraction #10.

There is now ~50 mL of 11:89 mixture in the graduated cylinder. Your goal is to raise its polarity to 12:88, and the table entry (under **EA to add**) says this requires another 0.5 mL of EA.

- Add 1/2 pipet load of EA to the mixture in the 100 mL graduated cylinder. Mix well.
- Transfer ~25 mL of this mixture to your column to collect fraction #11.

Only ~25 mL of 12:88 mixture remains in the graduated cylinder, and the table (under **EA to add**) says another 0.5 mL of EA will raise its polarity to 14:86.

- Add 1/2 pipet load of EA to the mixture in the 100 mL graduated cylinder. Mix well.
- Transfer ~25 mL of this mixture to your column to collect fraction #12.

In short, the table gives you a straightforward recipe for making sets of 4 fractions (#1-4, #5-8, #9-12, and so on). In each case, you begin by combining two solvents, EA and H, in the 100 mL of graduated cylinder to match the EA and H amounts given in **Starting EA:H** for the first fraction in the set (#1, #5, #9, …). The remaining 3 fractions in each set are created by adding a small volume of EA (use amounts given in **EA to add**) to whatever remains in the 100 mL graduated cylinder until you have collected 3 more fractions.

Remember that the ratios given under ** Nominal Composition** are just targets. You won’t hit them precisely, and you don’t need to. What you do need to do is to record in your notebook your procedure for making your fractions so that you (and any readers) can repeat your procedure if necessary.

A final note. If you ever need more than 24 fractions, use 40:60 EA:H to start fraction #25, and 50:50 EA:H to start fraction #29. Then construct the subsequent 3 fractions in each set by using a sequence of “add 3, add 3, then add 2 mL EA” (**EA to add**).