HPLC miniaturization

Since ISEs can be used in continuous flow systems or in flow systems with sample injection (flow injection analysis, FIA)21 their application is wide, not limited to discrete samples. Analysis time becomes shorter, with faster recycling. Additionally, in flow systems the experimental assembly and data analysis can be controlled automatically by microcomputer, including periodic calibration. Another development is the use of sensors for the detection of eluents of chromatographic columns in high-pressure liquid chromatography (HPLC). Miniaturization has permitted an increase in the use of sensors in foods, biological tissues, and clinical analyses in general. 

HPLC is extremely useful in monitoring and optimizing industrial processes. Conventional process monitors measure only bulk properties, such as the temperature and pressure of a reactor, while HPLC permits continuous realtime monitoring of consumption of starting materials, product composition, and impurity profile. There are a number of new initiatives relevant to HPLC for process monitoring, including sample preparation, automation, miniaturization, and specialized detectors. 

Because of its advantages (high sensitivity and selectivity, low cost and miniaturization) amperometric detection has been frequently used in flow injection analysis (FIA) and RP-HPLC. However, it has been established that the peak area (detector response) considerably depends on the flow rate. A general approach has been proposed to predict the effect of flow rate on the peak area in FIA and RP-HPLC. The general form of the correlation describing the flow in a parallel plate cell with short rectangular electrodes is... 

Micro-HPLC operation sets special demands on the gradient instrumentation. As the internal column diameter, d, decreases, lower flow rates should be used at comparable mean linear mobile phase velocities, u = 0.2-0.3 mm/s. At a constant operating pressure, the flow rate decreases proportionally to the second power of the column inner diameter, so that narrow-bore LC columns with 1mm i.d. require flow rates in the range of 30-100pL/min, micro-columns with i.d. 0.3-0.5mm, flow rates in between 1 and lOpL/min, and columns with 0.075-0.1 mm i.d. flow rates in the range of hundreds nL/min. Special miniaturized pump systems are required to deliver accurately mobile phase at very low flow rates in isocratic LC. 

Since the development of HPLC as a separation technique, considerable effort has been spent on the design and improvement of suitable detectors. The detector is perhaps the second-most important component of an HPLC system, after the column that performs the actual separation it would be pointless to perform any separation without some means of identifying the separated components. To this end, a number of analytical techniques have been employed to examine either samples taken from a fraction collector or the column effluent itself. Although many different physical principles have been examined for their potential as chromatography detectors, only four main types of detectors have obtained almost universal application, namely, ultraviolet (UV) absorbance, refractive index (RI), fluorescence, and conductivity detectors. Today, these detectors are used in about 80% of all separations. Newer varieties of detector such as the laser-induced fluorescence (LIE), electrochemical (EC), evaporative light scattering (ELS), and mass spectrometer (MS) detectors have been developed to meet the demands set by either specialized analyses or by miniaturization. 

Although this section provides a brief description of most commonly nsed detectors for HPLC, most of the focus is on a few detection modes. Optical absorbance detectors remain the most widely nsed for HPLC, and are discnssed in some detail. We also focns on flnorescence, condnctivity, and electrochemical detection, as these methods were not widely nsed for HPLC in the past, bnt are especially well suited to micro- and nano-flow instrnments becanse of their high sensitivity in small sample volumes. Mass spectrometry has also come into wide and rontine nse in the last decade, but as it is the subject of another chapter, it will not be fnrther discnssed here. Miniaturization has been particularly important for capillary and chip-based electrophoresis, which often employs sub-nanoliter detection volnmes [36,37]. 

The analysis time for chiral HPLC separations will probably remain relatively long until CSPs with higher efficiency than the present ones become available. But monolithic columns, columns with a smaller particle size (i.e., UPLC ), and miniaturized systems would increase the efficiency and speed up the enantioseparation of existing types of CSPs. 

Other innovations include PLE, MAE (see Section 1.3.1), and solid-phase microextraction (SPME). SPME is a sampling method applied to GC, HPLC, and CE. It is based on adsorbent- or adsorbent-type fibers and lends itself well to miniaturization. ... 

The most common analytical methods used were gas chromatography, HPLC, AA spectrophotometry, polarography, colorimetry, and potentiometry with ion-selective electrodes. In this study GC/MS and other more expensive instrumentation were avoided. If sorbent tubes could not be used for gaseous substances, then the less desirable miniature bubblers or impingers were considered. Although these devices are inconvenient they were often used because no better alternatives were available. Bags were used in a few cases where the analyte could not be retained on a sorbent because of volatility and a small tendency to sorb. Filters were used for particulates. Combinations of collection devices were used if we felt that both particulates and vapor might be present in the analyte. 

Miniaturization of HPLC-ICP-MS is an important issue in bioanalytical chemistry when small amounts of sample (e.g., single cells) need to be investigated.33 ICP-MS (with an octopole collision cell) in combination with nano-HPLC (75 pun column) was optimized for the detection of selenopeptides in a selenium-yeast protein digest after 100-fold preconcentration on a C18 capillary precolumn (300 (im column for salt removal and cleanup).34 Under identical separation and preconcentration conditions, electrospray MS/MS (using Nanospray qQqToF-MS - QSTAR from Applied... 

The RP-HPLC system was tested on the fractionation of OPPs and OCPs, from edible fats and oils of both animal and vegetal origin. When acetonitrile was used, the most polar OPPs eluted rapidly but tailed on this column, whereas the relatively nonpolar OCPs were retained the longest. Additional cleanup on miniature Florisil columns is required for the dirtier samples. A UV detector was used to determine the elution patterns of standards (78). The same system was used for the fractionation of OPP residues in processed bay foods prior to the final determination by GC without further cleanup but using a MS detector (66). 

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