Chromatography Forum

I was wondering if somebody could help me determine my max injection volume based on column dimensions. I am using a C18 column- 1.7um, 2.1mm x 100mm. It seems the solvent/MPA strength also plays a part in the injection volume so for reference:
MPA- 0.1% FA in H2O
MPB- 0.1% FA in ACN with a gradient elution starting at 30/70 MPA/MPB and ending at 100%B. My sample is being diluted first in 100% MeOH with the final dilution in 25/75 Water/ACN.

I have tried injecting 10uL and 20uL, and the peak shape still looks OK.

I have tried injecting 10uL and 20uL, and the peak shape still looks OK.

Proceed the same way until the peak shape (and N, Rs, response linearity...) looks not OK.

Capacity is limited by column geometry, but at what point is strictly a function of the partition coefficient...

If I remember correctly, we mainly used C18 column- 5um, 2.1mm x 100mm for most of our OTC-regulated/validated liquid products, extracting with methanol or DMF and using water/acetic acid/ACN isocratic mobile phases.

When we switched to 2.1mm columns from 4.6mm, I cut the injection volume to 5ul and we just kept it there as we had plenty of sensitivity.

Capacity is limited by column geometry, but at what point is strictly a function of the partition coefficient...

capacity of the column depends on the chemistry to much higher degree than column dimensions. In mixed-mode chromatography if have capacity which is 5-20 times higher than RP column. This also allows you to have a mismatch between diluent and mobile phase as well as the presence of salts in the sample and inject larger volumes. I just finished the study for bunch of columns for the analysis of the drug vildagliptin and compared Amaze SC mixed-mode column to leading brands RP column. Efficiency is twice higher on Amaze SC and peak shape is way better (1.5 in current gradient method vs. symmetry of 0.95-1 with isocratic methods on Amaze SC) for the same size particles, it also let inject compound in water (or high organic) and still not observe a peak disturbance on 50 uL injections.

This study is available upon request through our website.

Two things will figure in to the maximum amount you can inject.

First is concentration of the analyte. If you exceed the limit of the column with the amount of analyte you will see degradation of peak shapes.

The second is the composition of the sample matrix versus the Mobile Phase matrix. If you try to inject 100% Methanol into a MP that is mostly water you will have problems once the injection reaches a certain volume. If you exactly match your sample solvent matrix to the MP matrix then you are only limited by the first above, sample concentration.

Injecting a methanol matrix into a water or water/acetonitrile MP can cause what called Peak Surfing, where analytes that are more soluble in the methanol will tend to ride the methanol slug through the column if the injection volume is too large for the methanol to mix well with the MP. You can start small and increase injection volume until you see a peak coming out near the dead volume or solvent peak and that will determine your upper injection limit.

The best option if available is to match injection solvent to the MP at injection.

The max injection volume allowed for maintaining good peak shape depends on a number of factors:

1) Column dimension
2) Solvent strength of sample compared to mobile phase
3) Gradient or isocratic
4) Retention factor of the analyte of interest
5) analyte charge state
6) analyte conc

Sample column capacity (by volume and concentration) varies widely depending on the column's chemistry/bonding, mobile phase, injection solution, temperature and the SPECIFIC elution method. Max injection volume is often described using a general value, ~3 - 5% of the column volume. This range is a good starting value, but it is meaningless with testing. The only way it should be scientifically described is through running a proper loading study on your HPLC instrument. This is how we commonly do it for preparative HPLC applications, but also applies to analytical separations too.