Page 216 - Academic Press Encyclopedia of Physical Science and Technology 3rd Analytical Chemistry

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Encyclopedia of Physical Science and Technology EN008B-382 June 30, 2001 18:58

Liquid Chromatography 683

the capillary. In the low–pressure region, skimmers, oc-
topoles, and lenses concentrate and focus the analyte ions
into the ion trap which is an ion storage and mass anal-
ysis device. The ion trap permits MS/MS, and additional
n
molecular-structure information of MS can also be ob-
tained. The electrospray interface accommodates a broad
range of mobile phase compositions including volatile
buffers and 100% water at flow rates from 1–1000 µl/min.
A mass range up to 6000 m/z permits the analysis of some
protein and polymer samples.
On-line infrared and NMR detection are both possible
in conjunction with analytical or preparative HPLC. De-
spite the strong infrared absorbance of water and organic
solvents, the cylindrical internal reflection (CIRCLE) cell
with a cell volume of 24 µl offers a short pathlength al-
lowing for this background absorbance to be ratioed out.
Detection limits with the CIRCLE cell are in the low mi- FIGURE 11 Microbore chromatography with polarimetry detec-
cromolar range. Many NMR instruments can be fitted with tion (OA = optical activity). Chromatogram of a mixture contain-
a flow-through probe with a cell volume of about 120 µl. ing (1) injection peak, (2) l–2 octanol, (3) decane, (4) tetrade-
cane, and (5) hexadecane. Mobile phase: (A) (–)-2methyl–1–
Thestopped-flowmodemayberequiredtoallowforsignal
butanol in CH 3 CN (50 : 50) and (B) (±)-2methyl–1–butanol in
averagingtominimizethebackgroundduetoundeuterated CH 3 CN (50 : 50). Column = 1 mm × 25 cm 5-µm C-18 silica. Sam-
solvents. ple size = 0.5 µl; Flow rate = 20 µl/min. [From Bobbitt, D. R., and
A summary of the important parameters of the HPLC Yeung, E. S. (1984). Anal. Chem., 56, 1577. Reprinted with per-
detectors previously discussed is shown in Table II. The mission by the American Chemical Society.]
UV–VIS or PDA detector should be present on all HPLC
instruments for general application use. The other detec- value. Because of the reduced amount of packing ma-
tors should be added as demand for identification and anal- terial, sample sizes on the order of 0.2 to 1 µl are used.
ysis of certain classes of compounds arises. To prevent peak broadening, the volume of the flow cell
must be less than 2 µl and the dead volume caused by
fittings must be essentially eliminated. The advantages of
II. SMALL- AND LARGE-SCALE HPLC microbore HPLC are (1) a major decrease in solvent con-
sumption and hence cost, permitting more exotic solvents
The previous instrumental descriptionis typicalfor analyt- to be used, (2) a greater sensitivity when limited to a small
ical HPLC that can handle injected samples between 5 and sample, and (3) a potential for greater separation efficien-
100 µl. However, sometimes it is of interest for either very cies because of longer columns. A typical chromatogram
small or very large samples to be separated. Microbore is shown in Fig. 11.
and capillary HPLC can respectively analyze small and
even smaller samples while semipreparative and prepar-
B. Capillary HPLC
ative HPLC can respectively analyze large and larger
samples. Capillary LC has become more widely accepted as
commercial equipment to accommodate the low flow rates
of 1–5 µl/min, sample injection sizes of 60 nl, and the cap-
A. Microbore HPLC
illary detector flow cells has become available. Columns
The typical microbore HPLC column is 1 -in. tubing with generally 100–350 µmID × 25 cm in length packed with
16
an inside diameter of 1 to 2 mm and a length ranging from 3or5 µm particles are also commercially available. The
25 to 100 cm. Particle size of the packing material and main advantages of capillary LC are the small sample size
column-packing techniques are similar to those previously and improved sensitivity as compared to analytical or mi-
described. Because of the narrow column diameters, and crobore HPLC. Using the equation described in Section
longer lengths, flow rates on the order of 10 to 50 µl/min I.D, a 320-µm capillary could theoretically provide 200
are the norm. As shown in Table I, the plate count per unit times improvement in sensitivity as compared to a stan-
time and length are somewhat lower than with analytical dard 4.6-mm ID column assuming the same sample size
columns. However, these plates were achieved with a min- could be injected. However, for large volume injections
imal sacrifice in pressure drop as indicated by the N/psi with capillary LC (see Fig. 12), an on-column focusing