Roland W. Frei Hans J a m Udo A. Th. Brinkman Department of Analytical Chemistry Free University
De BOeleiaan 1083, 1081 HV Amsterdam The Netherlands
Postcolumn I REadion Ddectors for H P U High-performance liquid chromatography (HPLC) has become a powerful tool for analysis of a wide variety of samples. One of its shortcomings, however, is the often inadequate sensitivity and selectivity in the detection process, particularly when determining traces of compounds in complex matrices such as biological fluids or heavily contaminated environmental samples. Two possible ways to overcome these problems are the use of precolumn trace enrichment procedures which, by selective enrichment, also serve as a sample cleanup (I)and the use of chemical derivatization techniques which, based on the inherent selectivity and sensitivity of the chemistry used, will provide better methodology. Currently, the best and most reliable HPLC detectors are UV-VIS absorbance, fluorescence, and electrochemical detectors. It is attractive to try to expand their range of application by using suitable chemical derivatization techniques to convert the analytes of interest with their originally poor detection properties into compounds that can he detected with high sensitivity with these detectors. Besides an improvement of the detection properties, the chemical reaction can also enhance the selectivity of the total analytical method. The derivatization can be carried out either prior to the HPLC separation or by doing the reaction in an on-line postcolumn 0003-2700/85/A357-1529$01.50/0 @ 1985 American Chemical Societv
mode. Comparative advantages and disadvantages of these two approaches have been discussed previously (2). This paper will discuss on-line postcolumn derivatization. A general scheme of an HPLC system equipped with an on-line postcolumn reaction detector is given in Figure 1. Pump 1 is used for the eluent supply. After the compounds are separated on the analytical column, reagent is added with pump 2 via a suitable mixing device. The postcolumn reactor provides the desired holdup time for the reaction. Finally, the combined streams are passed through the detector. Several advantages of reaction detectors can he given, such as the following: Artifact formation plays a minor role in postcolumn reactors, as opposed to derivatization prior to the separation step. The reaction does not need to he complete and does not have to he fully defined. In addition, the reaction products need not he stable. The only requirement is reproducibility. * Different detection principles can he used in series; for example, a UV detector can he inserted directly at the HPLC column outlet, and a fluorescence or electrochemical detector can be inserted after the postcolumn reactor. The compounds are separated in their original form, which often permits adoption of separation procedures from the literature.
-
Pump 1
L @
Sampling vah/e
Analytical wiumn
T
Pump 2
Postcolumn reactor
1
Detector
Figure 1. General schematic of an HPLC system with reaction detectoi
ANALYTICAL CHEMISTRY. VOL. 57, NO. 14, DECEMBER 1985
1529 A
The disadvantages of postcolumn reaction detection are also quite obviOUS:
The need for postcolumn reagent addition requires an additional pump and possibly creates mixing problems. The optimum eluent for the separation is often not the optimum reaction medium; compromises are therefore necessary. Band broadening in the reactor can lead to serious losses in chromatographic resolution. Excess reagent can interfere with the signal of the reaction products. However. there are ways of reducing these disadvantages, which will be discussed. One obvious requirement is an optimum reactor design attuned to a particular reaction. In principle, three different types of reactors can be distinguished, the choice of which depends primarily on reaction kinetics: straight. coiled, or knitted open-tubu lar reactors (OTHs):packed-bed reac tors (PBRs); and segmented-stream tubular reactors (SSRs).As a rule of thumb, OTRs are recommended for fast reactions, PBRs for reactiona with intermediate kinetics, and SSRs for slow reactions. Band hroadening de. pends primarily on flow dynamic conditions in the reactor and can be predicted as far as OTRs and PBRs are concerned with the use of relatively simple equations. Huber et al. have published a good comparison between OTRs and PBRs (3).Special attention was given to the influence of the reactor on rhe loss of resolution and to the mixing process. Types ol reactors Open-tubular reactors. The postcolumn reactor that is simplest to construct is a piece of stainless steel or PTFE tubing. Coiling or knitting is attractive as it effects a relative decrease in band broadening (4.5). The dependence of band broadening in time units ( u , ) on various flow and geometry parameters is described by the well-known equation:
In this equation, n(O < ~51) accounts for the reduced band broadening for coiled or knitted tubes as compared with straight ( n = 1) tubes, t , is the mean residence time in the reactor, dt is the tube inner diameter, and D, is the molecular diffusion coefficient. From this equation, it can be concluded that smaller internal diameter tubes lead to lower band broadening. On the other hand, pressure drop, Ap, over the reactor increases with decreasing internal diameter, as expressed by the Poiseuille equation: 1530A * ANALYTICAL CHEMISTRY, VOC.
om
Arnlno acas
PBR
F F
Barbfitrates
Norescta uv
carbamates
OTR
Catecholamines Chlwoanilines
SSR F OTR.PBR, F and SSR om F
Ap =
F
PBR SSR
1 F
OTR
E
18
2cyanalcetamide
om
F
17
Variarsreagems
OTR
WendVIS
18
F
la
512qF2t, r2d,6
(2)
where n is the viscosity of the reactor fluid and F is the total volumetric flow rate. As a rule of thumb, open-tubular reactors are best suited for fast reactions up to about 30 3. Knitted open-tubular reactors may be useful for reactions of several minutes, according to Engelhardt (4). Typical reactions suitable for open-tubular reactors are the well-known fluorogenic reactions with fluorescamine (Fluram) and o-phtbalaldehyde (OPA).Both reagents react rapidly with primary amines. Packed-bed reactors. Packed-bed reactors are glass or stainless steel columns that are packed with small, inert, nonporous glass beads. These reactors can be considered as HPLC columns operated under nonretention conditions. Band broadening can therefore be given by the equation:
in which h is the reduced plate height (with values of between two and about six, mainly dependent on the packing quality), d, is the particle diameter, and L is the reactor length. From this equation, it follows that reducing the panicle size or increasing the length at constant residence time causes a reduction in band broadening. However, pressure drop over the reactor is related to the same parameters:
where KOrepresents the permeability 57, NO. 14. DECEMBER 1985
constant, which has a value of 0.001-0.002. The use of packed-ma reactors is usually indicated for intermediate reaction times of about 0.54.0 min. Segmented-stream tubular reactors. For slower reactions and also as an alternative to bed reactors segmented systems can be used. This technique is based on segmenting the column effluent with air bubbles or nonmiscible solvent plugs. If no leakage from one segment to another occurs and if the segment size is sufficiently small, axial dispersion and thus band broadening are efficiently reduced. Suppression of leakage can be achieved by a proper choice of the reactor material (6). The theoretical treatment is rather complex. Generally speaking, the total band broadening of the postcolumn system is largely determined by the Tpiece and the phase separator or debubbler, needed to create a homogeneous flow through the detector, rather than the postcolumn reactor itself. Segmented-stream reactors are useful for slower reactions (reaction times up to about 20 min) and when two-phase reactions or extractions are carried out. Application of postcolumn reaction detection. In Table I some typical applications of postcolumn reaction detectors have been summarized. This table is by no means complete and is intended only to give an idea of the broad range of possibilities of this technique. Examples of the use of the different reactor types and detection modes are collected in this table. More applications can be found, for example, in Reference 7, which is also recommended for additional reading.
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