The InLine Flow Sensor

The alternative to a transport interface is an in-line flow-through cell, and in 1983 a micro IR cell, 3.2 |L in volume, that fitted directly into the IR spectrometer was described by Brown and Taylor. By using a small-bore column they achieved an overall increase in mass sensitivity of about two orders of magnitude, relative to that obtained from the standard 4.6 mm i.d. column. An FTIR spectrometer was used, but the actual sensitivity improvement was confused as the length of the small bore column differed significantly from that of the standard column. Consequently, the true sensitivity in terms of minimum sample mass that would provide an acceptable spectrum, could not be assessed accurately.

A different cell design cell for use with LC microbore columns interfaced with an FTIR spectrometer

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Wavenumbers (cm 1)

Figure 5 A three-dimensional reversed phase LC/IR plot of the separation of some polynuclear aromatic hydrocarbons. (Reproduced with permission from Conroy and Griffiths, 1984.)

1400 1200 1000 800

Wavenumbers (cm 1)

Figure 5 A three-dimensional reversed phase LC/IR plot of the separation of some polynuclear aromatic hydrocarbons. (Reproduced with permission from Conroy and Griffiths, 1984.)

was described by Johnson and Taylor. It was claimed that the cell would reduce the detection limit (the minimum mass required to produce a useful IR spectrum) to about 50 ng. The flow cell design is shown in Figure 6.

The cell was formed crystalline calcium fluoride or potassium bromide in the form of a block 10x10x6 mm. A hole 0.75 mm i.d. was drilled through it to carry the mobile phase from the column through the block and out to waste. The collimated IR beam passed through the block, normal to the cylindrical aperture and, in doing so, transversed a section of the exiting eluent. A beam condenser was used to reduce the focal diameter of the beam to that of the hole. It was noted that the maximum signal-to-noise was obtained by summing the spectra from scans taken across the peak, between + 1.53 a of the Gaussian profile, as it passed through the cell. As a practical point of interest, it was found easier to modify optically the size of the IR beam to match the flow cell, than to construct a cell that would accurately match the dimensions of the IR beam.

Sabo et al. developed an attenuated total reflectance cell for both normal- and reversed-phase

Figure 6 Zero dead volume micro IR cell.

chromatography. The cell was made with cone shaped ends, from a cylindrical shaped zinc selenide crystal, and mounted in stainless steel. The crystal was blazed at 45° and consequently gave ten reflections during passage of the IR beam down its length. The incident beam was focused onto the cone face and the radiation leaving the crystal was focused onto the IR sensor. The cell volume was large, ca. 24 |L and thus would adversely affect the resolution of a small-bore column. Clear, identifiable spectra were obtained from a 100 |L sample, containing 2% of acetophenone and ethyl benzoate and 1% of nitrobenzene from on-the-fly spectra. However, this was not a very sensitive device compared with other LC/FTIR systems.

A rather complicated solvent extraction system was developed by Conroy and Griffiths for use with an LC/FTIR tandem instrument. It involved a process that continually extracted the solute from the column eluent into dichloromethane. The solution is dichloro-methane was concentrated and dispersed onto a plug of potassium chloride powder. The residual solvent was evaporated, the sample scanned and a spectrum taken. This process is somewhat clumsy but it introduces a new concept for constructing LC/IR interfaces. Employing the same basic principle Johnson et al. constructed a rather unique extraction cell for use with an LC/IR tandem system by introducing the technique of segmented flow. The aqueous eluent from a reversed phase column was mixed with chloroform (with which the column eluent was immiscible) producing segmented flow. The extraction solvent (chloroform) was then separated from the segmented flow by means of a 'hydrophobic' (dispersive) membrane. There were two pumps, one for the mobile phase and the other for the extraction solvent, which could be either chloroform or carbon tetrachloride. The two streams were mixed at a T junction (post column) and formed the segmented flow. The segmented flow then passed through an extraction coil and then to a separator. The separator was made of stainless steel with a membrane having pores about 0.2 | m in diameter dividing its length into half and its general layout is shown in Figure 7.

The volume on either side of the membrane was about 16 |L and the amount of solvent passing through the membrane was controlled by the differential pressure across the membrane. Obviously this device could cause serious peak dispersion and would be unsuitable for use with high-efficiency of smallbore columns. Samples containing at least 300 |g of material were necessary to produce a satisfactory spectrum, indicating a relatively poor sensitivity.

The segmented flow interface was developed further by Hellgeth and Taylor, who improved both the

Membrane Figure 7 An extraction interface for LC/IR.

segmentation and the extraction efficiency. The segmented flow generator was made from 1/16 in. Swagelok T union, drilled out to contain 1/16 in. tubes the ends of which were only 0.45 mm apart and the general design is shown in Figure 8.

The column eluent and extraction solvent passed into the mixing T through tubes 0.020 in. i.d. The exiting segmented flow passed through an extraction conduit consisting of a Teflon™ tube, 75 cm long and 0.8 mm i.d. The membrane separator was constructed from two stainless steel plates with grooves in each surface, and a triple-layer membrane of Gore-TexTM sheet. The membrane was made from two materials. The inner layer comprised an unsupported 1 |im pore Teflon™ sheet which was sandwiched between two outer sheets of 1 |im pore Teflon™. These sheets were supported by non-woven polypropylene membranes which were located on the outer surfaces. The infrared cell was a modified Spectra-Tech Inc. demountable flow cell fitted with windows of either calcium fluoride or zinc selenide. The system appeared to function reasonably well; satisfactory spectra were obtained from 100 |g of material. Although a considerable improvement, the sensitivity was still relatively poor compared with that obtained with the rotating disc transport interfaces.

Further work by Somsen et al. has resulted in a segmented flow concentrator with significantly reduced band dispersion. A conventional liquid chromatograph was employed. It included a pump, pulse damper, injection valve and column. The column eluent entered a T piece where it was joined by an immiscible extraction solvent, usually methylene dichloride, supplied from another pump and pulse damper.

Figure 8 Diagram of a phase separator.

The extraction solvent flowed through a second column situated prior to the T piece to provide more pulse damping. The segmented mixture then passed through an extraction coil which provided the necessary time for the solutes to diffuse from the aqueous phase into the solvent. There is no parabolic velocity profile in segmented flow, and thus little or no peak dispersion can occur.

The segmented flow entered a phase separator and the separated solvent then passed through a UV absorption detector and into a spray jet assembly. Heated nitrogen was used in the spray jet assembly, to aid in the nebulization process. The chromato-grams obtained indicate that very little peak dispersion occurs and that the column resolution is not significantly degraded. The finite volume of the extraction tube, however, produced a significant retention delay (about 3.5 min), which varied with both the flow rate and volume of the extraction system. Providing the solvents were reasonably volatile, they were completely removed in the nebulizing process. However, the percentage of organic solvent in the mobile phase must not be large enough to make it miscible with the methylene dichloride and prevent the formation of segmented flow. It follows that the choice of mobile phase was somewhat restricted.

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