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0 INTRODUCTION
High performance liquid chromatography is basically a highly improved form of
column chromatography. Instead of a solvent being allowed to drip through a column under
gravity, it is forced through under high pressures of up to 400 atmospheres. That makes it
much faster.
It also allows to use a very much smaller particle size for the column packing material
which gives a much greater surface area for interactions between the stationary phase and
the molecules flowing past it. This allows a much better separation of the components of the
mixture.
The other major improvement over column chromatography concerns the detection
methods which can be used. These methods are highly automated and extremely sensitive.
Typical HPLC system consists of followings (Figure 1).
1.174, 0.1211,
1 and 2 1.68
1.376 0.1189
4.079, 0.1189,
2 and 3 17.99
1.376 0.1816
1
7.064, 0.1816,
3 and 4 13.77
4.079 0.2519
26.479, 0.8178,
4 and 5 36.30
7.064 0.2519
1.242, 0.0977,
1 0.82
1.151 0.1242
1 and 2 0.86
1.251, 0.0973,
2 0.89
1.151 0.1270
2.132, 2.137,
1 7.59
1.242 1.251
2 and 3 7.58
0.1368, 0.1367,
2 7.57
0.977 0.0973
2.864, 0.1694,
1 4.78
2.132 0.1368
3 and 4 4.80
2.872, 0.1690,
2 4.81
2.137 0.1367
6.393, 0.4076,
1 12.23
2.864 0.1694
4 and 5 12.25
6.403, 0.4068,
2 12.27
2.872 0.1690
b) Effect of the gradient elution
2( tR2 − tR1 )
Resolution, Rs = 𝑊𝑏1+𝑊𝑏2
2(1.251−1.151)
Rs(12) =
0.1270+0.0973
= 0.90
2(1.292−1.161)
Rs(12) =
1.1029+0.0907
= 1.32
4.0 DISCUSSION
The peak order is caffeine followed by acetone, methyl benzoate, phenatole and
phenanthrene.
6.0 REFERENCES