Inject a 20 m l aliquot onto a 15 cm 46 mm column

Inject a 20- m L aliquot onto a 15-cm 4.6-mm column packed with a 5
m m C8-bonded stationary phase. The isocratic mobile phase is 37.5:62.5
v/v acetonitrile and water (containing 1.5 g of tetramethylammonium
perchlorate and 0.1 mL of 70% v/v HClO
4
). Monitor the chromatogram
using a fluorescence detector set to an excitation wavelength of 235 nm
and an emission wavelength of 310 nm.
Q
UESTIONS
1. The solid-phase extraction is important because it removes constitu-
tions in the serum that might interfere with the analysis. What types
of interferences are possible?
Blood serum, which is a complex mixture of compounds, is approxi-
mately 92% water, 6–8% soluble proteins, and less than 1% each
of various salts, lipids, and glucose. A direct injection of serum is
not advisable for three reasons. First, any particulate materials in the
serum will clog the column and restrict the flow of mobile phase. Sec-
ond, some of the compounds in the serum may absorb too strongly
to the stationary phase, degrading the column’s performance. Finally,
although an HPLC is capable of separating and analyzing complex
mixtures, an analysis may still be difficult if the number of constitu-
ents exceeds the column’s peak capacity.
2. One advantage of an HPLC analysis is that a loop injector often
eliminates the need for an internal standard. Why is an internal stan-
dard used in this analysis? What assumption(s) must we make when
using the internal standard?
The best way to appreciate the theoretical
and practical details discussed in this sec-
tion is to carefully examine a typical ana-
lytical method. Although each method is
unique, the following description of the
determination of fluoxetine in serum pro-
vides an instructive example of a typical
procedure. The description here is based
on Smyth, W. F. Analytical Chemistry of
Complex Matricies , Wiley Teubner: Chich-
ester, England, 1996, pp. 187–189.
For a review of solid-phase extraction
(SPE), see Section 7F.5. Table 7.8 de-
scribes the properties of several different
types of SPE cartridges. Figure 7.22 shows
a photo of SPE cartridges, and Figure 7.23
illustrates the steps in completing a solid-
phase extraction.

843
Chapter 12 Chromatography and Electrophoresis
12E.6 Evaluation
With a few exceptions, the scale of operation, accuracy, precision, sensitivity,
selectivity, analysis time, and cost for an HPLC method are similar to GC
methods. Injection volumes for an HPLC method are usually larger than
for a GC method because HPLC columns have a greater capacity. Because
it uses a loop injection, the precision of an HPLC method is often better.
HPLC is not limited to volatile analytes, which means that we can analyze
a broader range of compounds. Capillary GC columns, on the other hand,
have more theoretical plates, and can separate more complex mixtures.