Instructions for UV/Vis sample preparation: dilution

In order to analyze concentrations with a UV/Vis spectrophotometer, it is necessary to

have your solution concentration such that the absorbance is between about 0.05 to
about 1.0. Absorbance is dependent on concentration according to Beer's Law

A is absorbance,
A = ε·c·l ε is the extinction coefficient (1/concentration/pathlength)
c is concentration in units of concentration
l is the pathlength of the cuvette

optically clear window

for light beam
transmission
1 cm
Notice this equation is linear, with no intercept, meaning that at a concentration of zero,
a solution has an absorbance of zero. This equation should hold for the absorbance
measurement to be meaningful.

Example: if a compound has an extinction coefficient of 8800 lit/mmol/cm, what is the

range of concentrations you may use to determine an unknown solution concentration?

Absmax = 1.0 = 8800 (lit/mmol/cm)· cmax(mmol/lit) · 1 cm

cmax = _______________

Absmin = 0.05 = 8800 (lit/mmol/cm)· cmin(mmol/lit) · 1 cm

cmin = _______________

since it is an unknown concentration, it is difficult to know whether it is in the range of

cmin < c < cmax

Thus, the proper range of concentrations is identified by simply diluting the solution until
it has an absorbance range

Absmax < Abs < Absmin

How much should you dilute your solution?

If you knew that in advance, this process would be simple. Unfortunately it is trial and
error until you have experience with a specific solution in a specific process.

When you have absolutely no idea where to start, the best approach is to make serial
dilutions.
Serial dilutions are made by dilution your solution sequentially such as:

dilute sample 1/10 to make your first dilution

dilute your first dilution 1/10 to make your second dilution
dilute your second dilution to make your third dilution
and so forth. Then the dilution factors for your solutions are

sample
first dilution x 101 = sample concentration
second dilution x 102 = sample concentration
third dilution x 103 = sample concentration

Measure the absorbance of each of your dilutions until the value falls in the proper
range.

Note that the highest value you will measure with the spec is around 3, and that could
be 3, 30, 300 or more...

Finally, once you have found that magic dilution that falls in range, you should report

Absorbance (you measured this), dilution factor, unknown solution concentration

The value of the absorbance you measured does not have meaning to the reader of your
report. What they care about is the actual concentration of the solution, so be sure to be
complete in your presentation of your data and finish the calculation WITH ERROR
estimate.

Measurement of Optical Density

Optical density is an indirect measurement of the amount of bacteria in a solution.

Bacteria are small particles and as such, they don't absorb light but the block it. Because
this is a different phenomena than absorbance, the concentration range for the
measurement of optical densities is about 0.05 to 0.50. You need to dilute your bacteria
solution to have an "absorbance" reading between those values to be accurate.

Note about optical densities

1) bacteria settle quickly so you should mix the solutions well just prior to pipetting them.
2) you can remix the bacteria in the cuvette by pipetting carefully up and down just prior
to putting the cuvette into the spectrophotometer.
3) bacteria may lyse in water solutions, so the optical density reading may change over
time.