Page 232 - 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 699

reaction conditions are flexible. Another advantage is that
small poorly retained molecules can be modified to more
hydrophobic, longer retained compounds. The disadvan-
tage is that sample preparation time is increased and au-
tomation is hampered. Because postcolumn derivatization
involves mixing the column effluent with the reagent to
form the derivative just before it passes into the detector,
this method is compatible with automation. Generally, a
low-pressure reagent pump, a connecting tee, and a mixing
coil are all that are required beyond the basic HPLC instru-
ment. One further advantage of postcolumn derivatization
is that the formation of a stable or chromatographically
separable product is not necessary. However, fairly strin-
gent conditions are necessary for postcolumn derivatiza-
tion. These are

1. Reaction must be rapid, within about 2 minutes, and
reproducible. FIGURE 29 Separation of OPA amino acid derivatives on a 5-µm
2. The reagent volume should be small relative to the 25-cm C-18 column. Mobile phase: citrate/phosphate buffer, pH
eluent volume to minimize band broadening. 7.7 with convex methanol gradient from 20 to 70% methanol
in 15 min. Fluorometer excitation λ = 330 nm; emission λ = 418
3. The reaction should be a simple one step procedure to
nm cutoff filter. Peaks: (1) cysteic acid, (2) Asp, (3) Glu, (4) S-
minimize additional hardware needs. carboxymethyl cystein, (5) Asn, (6) Ser, (7) Glu, (8) His, (9) me-
4. The reagent itself must have none or a very low thionine sulfone, (10) Thr, (11) Gly, (12) Arg, (13) β-Ala, (14) Tyr,
detector response. (15) Ala, (16) α-aminobutyric acid, (17) Trp, (18) Met, (19) Val, (20)
Phe, (21) NH , (22) IDE, (23) Leu, (24) Orn, (25) Lys. [From Lin-
+
4
droth, P., and Mopper, K. (1979). Anal. Chem., 51, 1668. Reprinted
Most of the HPLC derivatization chemistry is based with permission by the American Chemical Society.]
on well-known reactions reported in organic or inorganic
chemistry research. For example, the fluorescent deriva- from the nitrogen sheath gas. Steroids, amino acids, or
tization of primary amines such as amino acids using o- DNA as pentafluorobenzyl derivatives can act as electron
phthalaldehyde (OPA) and mercaptoethanol either in the capturing compounds to generate negative ions. Detection
precolumn or postcolumn mode has been well studied. limits are improved 100 times to attomole levels.
The use of enzymes as precolumn and post-column
CHO modification reagents for HPLC has been investigated.
pH 10
SCH 2 CH 2 OH RNH 2 For example, nucleoside phosphorylase will catalyze the
reaction of orthophosphate and inosine to form hypox-
CHO
anthine and ribose–6–phosphate. After running the en-
zyme reaction, separation of the hypoxanthine formed
SCH 2 CH 2 OH
from the substrate inosine by HPLC permits the deter-
mination of phosphate in complex matrices. Creatine ki-
NR
nase (CK) isozymes have been separated by HPLC and
detected using the luciferase bioluminescence reaction.
Scheme 1 CK
creatine phosphate + ADP creatine + ADP

A typical chromatogram of OPA amino acid derivatives
luciferase
at the nanogram level is shown in Fig. 29. Dansylation ATP + luciferin
reactions to form fluorescent derivatives of amines or car-
adenyl luciferin + pyrophosphate + light,
bonyl compounds have also been applied to HPLC. Com-
plexation of transition metals with pyridylazoresorcinol where ADP and ATP are adenosine-diphosphate and
(PAR) either in the precolumn or postcolumn mode has -triphosphate, respectively.
been a popular method. Precolumn derivatization has been Postcolumn addition of the substrates of both enzymes
shown by Ian Blair and co-workers to be useful even for and luciferase into a flow cell mounted adjacent to a pho-
LC/MS. Atmospheric pressure chemical ionization com- tomultiplier tube provided sensitive and specific detection
monly used in LC/MS can provide a source of electrons of the CK–MB and CK–BB isoenzymes in serum samples.