high performance liquid chromatography, broad peaks

Injection volume in HPLC

Injection volume

Injecting samples into the HPLC injector is very simple; this is done by manual syringe or auto-sampler injection. 

The Rheodyne sample loops and auto-sampler have fixed or variable volumes (0.1 – 100 μL) that can inject into the HPLC column.

The peak area and height increase in proportion to the injection volume of the sample solution. When the injection volume becomes too high the result in the peak is broadened and broadening this peak may reduce the resolution between adjacent peaks.

The injection volume must be chosen in accord with the column geometry. We can use a volume between 1 and 5% of the column dead volume as the maximum injection load per column; however, we have to determine this.

The best injection volumes are related to the volume of the HPLC column and, as a result, are dependent on the column length and cross-sectional area.Here some approximate volume range of the sample component is mentioned if it is prepared in the same mobile phase as used in the system.

Column ID
Length of Column
Injection Volume
2.1 mm
30 to 100 mm
1 to 3 µL
3.0 to 3.2 mm
50 to 150 mm
2 to12 µL
4.6 mm
50 to 150 mm
8 to 40 µL
10 mm
50 to 250 mm
40 to 100 µL
21.2 mm
50 to 250 mm
150 to 300 µL
30 mm
50 to 250 mm
300 to 700 µL
50 mm
50 to 250 mm
1000 to 2000 µL

A. Wikipedia: https://en.wikipedia.org/wiki/High-performance_liquid_chromatography

B. Knauer: https://www.knauer.net/en/search?q=chromatography

C. Kromasil: https://www.kromasil.com/support/faq.php

D. Shimadzu: https://www.shimadzu.com/an/service-support/technical-support/analysis-basics/basic/what_is_hplc.html

E. ChemistryView: https://www.chemistryviews.org/details/education/9464911/What_is_HPLC/


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