Conceptually, What's Going On?
Let's think on a conceptual basis about what is going on with the various examples of Figure 1. If the sample is injected
with mobile phase as the injection solvent, it does not have to undergo any chemical changes, so analyte molecules should
immediately begin to interact with the stationary phase in the column and undergo normal retention behavior. However, if the
injection volume is large enough, you can imagine that some band broadening will take place during the injection. For example,
if the injection is extremely large, such as 1 mL, the first sample molecules will travel a significant way through the column
before the last of the sample molecules arrive at the inlet. The distance between the first and last sample molecules in a
given band determine the peak width, so a broad peak would be observed. At the other extreme, an infinitely small injection
would have none of this injection-related broadening, so the peak would be much narrower. As we'll see later, we generally
can tolerate an injection volume of approximately 15% of the peak volume of the first peak of interest before we find the
injection-related band broadening objectionable.
When the injection solvent is changed, we have another variable in addition to the injection volume mentioned above. In this
case, the injected solvent plug must be diluted to the same composition as the mobile phase before "normal" retention can
occur. If we think of the injection plug as a ball of solvent at the inlet of the column, it will shrink as it travels through
the column, with the outside edges being diluted to the mobile-phase concentration before the center of the ball. While they
are inside the ball of the injection solvent, molecules will travel down the column as if that solvent was the mobile phase,
whereas those diluted into the mobile phase will now travel at the normal rate in the mobile phase. So if the injection solvent
is stronger than the mobile phase, it will tend to sweep analyte molecules through the column more quickly than normal. Because
some analyte molecules travel more quickly (in the middle of the injection plug) than others (on the outer edges), injection
in a solvent stronger than the mobile phase tends to smear the sample along the column, causing peak distortion. On the other
hand, if the injection solvent is weaker than the mobile phase, analyte molecules inside the injection plug will move more
slowly than normal, which tends to concentrate them at the column inlet. This often is referred to as on-column concentration
and is a technique that can be used to inject large volumes of dilute samples without excessive band broadening. And, as you
might expect, the influence of the injection solvent depends on both its volume and how much its composition differs from
the mobile phase. A small volume of a strong solvent will be less detrimental than a large volume of the same injection solvent,
and at a small enough volume, even a very strong injection solvent will get diluted very quickly, so it will be of little
How Much Is Too Much?
It can be shown (see discussion of reference 2) that if you want to have <1% loss in resolution because of injection-related
peak broadening, the injection volume should be <15% of the peak volume of the first peak of interest if mobile phase is used
as the injection solvent. If you are willing to tolerate a 10% loss in resolution, you can increase this to <40% of the peak
volume. You can determine the peak volume of a peak by measuring its baseline width (or half-height width × 1.7) and multiplying
by the flow rate. Alternatively, you can get a good estimate of the peak volume with a few simple calculations that follow.
We think it is a good idea to make this calculation and compare the results with the actual measurements as a double-check
to make sure there isn't excessive peak broadening because of other causes, such as a bad column.
We can use the data of Figure 1 as an example. The expected width can be estimated from the column plate number, N:
R is the retention time and w is the peak width at baseline between tangents drawn to the sides of the peak. Equation 1 can be rearranged to
Now we need a value of N, which for real samples under practical separation conditions can be estimated as
where L is the column length (in millimeters) and d
p is the particle diameter (in micrometers). The column of Figure 1 is 250 mm long packed with 10-μm particles, so N ≈ 7500. Injected in the weakest solvent, water (Figure 1d), retention times are 4.72 and 9.05 min. With equation 2, this
translates to expected peak widths of 0.218 and 0.418 min, respectively. (The usual disclaimer applies here: Values have been
rounded for convenience, so if you try to reproduce these calculations, your results may vary somewhat.) Multiply by the flow
rate (1 mL/min) to convert this to peak volumes of 218 and 415 μL.
If we use the <15% rule mentioned at the beginning of this section, maximum recommended injection volumes in mobile phase
are then 0.15 × 218 = 33 μL and 0.15 × 418 = 63 μL. If we are interested in the first peak, this will be the limiting case,
so injection of 5–30 μL in mobile phase would be expected to cause less than 1% loss in resolution. If we are more liberal
with the tolerance for loss of resolution, as might be justified in the case of Figure 1, where resolution >>2, the <40% rule
should be adequate, or 0.4 × 218 = 87 μL. Similar calculations can be performed for the data of Table I and we would see that
the <15% rule would allow approximately 45-μL injections and the <40% rule would allow approximately 120-μL injections.