Absorbance minimum detectable

Path length (m) Absorbence Minimum Detectable Concentration (ppm) " (20 metre cell)... 

It is instructive to compare the sensitivity which may be achieved by absorption and fluorescence methods. The overall precision with which absorbance can be measured is certainly not better than 0.001 units using a 1 cm cell. Since for most molecules the value of emax is rarely greater than 105, then on the basis of the Beer-Lambert Law the minimum detectable concentration is given by cmin> 10 3/105= 10 8M. 

EXAMPLE 1.5 The sensitivity of luminescence. Consider a photoluminescence experiment in which the excitation source provides a power of 100 ptW at a wavelength of400 nm. The phosphor sample can absorb light at this wavelength and emit light with a quantum efficiency of r] = O.I. Assuming that kg = 10 fii.e., only one-thousandth of the emitted light reaches the detector) and a minimum detectable intensity of l(f photons per second, determine the minimum optical density that can be detected by luminescence. 

Narrow-bore columns of between 1.0 and 2.5 mm ID are available for use in specially designed liquid chromatographs having an extremely low extracolumn dispersion. For a concentration-sensitive detector such as the absorbance detector, the signal is proportional to the instantaneous concentration of the analytes in the flow cell. Peaks elute from narrow-bore columns in much smaller volumes compared to those from standard-bore columns. Consequently, because of the higher analyte concentrations in the flow cell, the use of narrow-bore columns enhances detector sensitivity. The minimum detectable mass is directly proportional to the square of the column radius (107) therefore, in theory, a 2.1-mm-ID column will provide a mass sensitivity about five times greater than that of a 4.6-mm-ID column of the same length. 

Two UV detectors are also available from Laboratory Data Control, the UV Monitor and the Duo Monitor. The UV Monitor (Fig.3.45) consists of an optical unit anda control unit. The optical unit contains the UV source (low-pressure mercury lamp), sample, reference cells and photodetector. The control unit is connected by cable to the optical unit and may be located at a distance of up to 25 ft. The dual quartz flow cells (path-length, 10 mm diameter, 1 mm) each have a capacity of 8 (i 1. Double-beam linear-absorbance measurements may be made at either 254 nm or 280 nm. The absorbance ranges vary from 0.01 to 0.64 optical density units full scale (ODFS). The minimum detectable absorbance (equivalent to the noise) is 0.001 optical density units (OD). The drift of the photometer is usually less than 0.002 OD/h. With this system, it is possible to monitor continuously and quantitatively the absorbance at 254 or 280 nm of one liquid stream or the differential absorbance between two streams. The absorbance readout is linear and is directly related to the concentration in accordance with Beer s law. In the 280 nm mode, the 254-nm light is converted by a phosphor into a band with a maximum at 280 nm. This light is then passed to a photodetector which is sensitized for a response at 280 nm. The Duo Monitor (Fig.3.46) is a dual-wavelength continuous-flow detector with which effluents can be monitored simultaneously at 254 nm and 280 nm. The system consists of two modules, and the principle of operation is based on a modification of the 280-nm conversion kit for the UV Monitor. Light of 254-nm wavelength from a low-pressure mercury lamp is partially converted by the phosphor into a band at 280 nm. 

Very recently, Bailey and Richards (23) have shown that a high degree of sensitivity for adsorbed species can be achieved by measuring the absorption of infrared radiation on a thin sample cooled to liquid helium temperature. The optical arrangement used in these studies is shown in Figure 10. The modulated beam produced by the interferometer is introduced into the UHV sample chamber and reflected off a thin slice of monocrystalline alumina covered on one side by a 1000 k film of nickel or copper. Radiation absorbed by the sample is detected by a doped germanium resistance thermometer. The minimum absorbed power detected by this device when operated at liquid helium temperature is 5 x 10 14 W for a 1 Hz band width. With this sensitivity absorbtivities of 10"4 could be measured. 

As a result of the almost perfect correlation of the spectral intensity values within the small range of observation, the minimum detectable absorbance signal is determined only by statistical variations of the intensity between the neighboring pixels (shot noise). This means that an increase in radiation intensity or of the measurement time by a factor of 4 will reduce the absorbance noise by a factor of 2 (square root of 4). 

Diltiazem hydrochloride and its related compounds can be separated in both bulk drug and finished tablets using a Waters pBondapak C18 column (10 pm particle size, 300 mm x 3.9 mm I.D.) and a mobile phase of buffer methanol acetonitrile (50 25 25, v/v) at a flow rate of about 1.6 mL/minute. The buffer is 0.1 M aqueous sodium acetate containing 5mM d-camphorsulfonic acid (99%) adjusted to pH 6.2 with 0.1 M aqueous sodium hydroxide. Detection of the compounds is achieved using UV absorbance at 240 nm. The method provides for the resolution of trans-diltiazem and seven known and unidentified related compounds. Diltiazem hydrochloride elutes at approximately 21 minutes under these conditions. The minimum detectable amounts are less than 0.1% for all related compounds except for one of the synthetic intermediates for which there is a limit of about 2% (27). 

Several reversed-phase methods were also developed which do not use a C18 column. A reversed-phase method using a C8 Spherisorb column has been reported (54) to quantitate diltiazem and two of its metabolites (N-monodemethyl diltiazem and desacetyl diltiazem). A 10 pm particle size PRP-1 column (55), mobile phase of 60% acetonitrile and 0.01 M aqueous KH2PO4, 40% 0.005M aqueous tetrabutylammonium hydroxide and UV absorbance detection at 254 nm was used to determine diltiazem present in plasma. Several HPLC methods have been developed which use a cyano-bonded column. One such method was developed for the determination of diltiazem and its metabolite desacetyl diltiazem in human plasma (56). The analytes are extracted from plasma made basic with 0.5M aqueous dibasic sodium phosphate (pH 7.4) using 1% 2-propanol in hexane. The method uses a cyanopropylsilane column with a mobile phase of 45% acetonitrile and 55% 0.05M aqueous acetate buffer (pH 4.0). The minimum detectable limit was 2 ng/mL in plasma. A similar HPLC method was developed by Johnson and Pieper (57) for the determination of diltiazem and three of its metabolites. Also, an HPLC method was developed (58) for the analysis of diltiazem and desacetyl diltiazem in plasma using UV detection at 237 nm, a Zorbax CN 6 pm particle size column and a mobile phase of 45% methanol, 55% 0.05M aqueous ammonium dihydrogen phosphate and 0.25% triethylamine adjusted to pH 5. 

In the case of the detection limit we have to distinguish between the minimum detectable concentration and the minimum detectable mass. The minimum detectable concentration of a solute in the sample solution depends only on the detector properties and on the optical properties of the solute, i.e. its absorbance, if the maximum tolerable sample volume with respect to the retention volume of this solute is injected. The minimum detectable concentration is independent if the column dimensions, plate number or capacity factor. 

A similar technique for the determination of water in isopentane was developed by Baranova et al. [65]. Water was absorbed in a trap containing triethylene glycol at 25°C, then desorbed at 100°C. The minimum detectable water concentration was 1 10 %. 

Null-Background Techniques. In conventional absorption spectroscopy the difference between two laige quantities, the incident and transmitted intensities, is measured, thus limiting the minimum detectable absorbance, 4 to lO-3. This can be greatly improved using null-background... 

The UV spectrum of palytoxin, as discussed earlier, shows two characteristic absorption peaks at 233 and 263 nm, contributed by the respective chromophores (Figure 29.4). The ratio of their absorbance (233 versus 263 mn), which is approximately 1.7 [3], is characteristic and indicative of the toxin s presence. The absorptions at either wavelength have been reported to be linearly related to palytoxin concentration in the range of 5-20 pg/mL. However, the disadvantage of this method is its detection limit, as the minimum detectable concentration has been reported to be 5 pg/mL (palytoxin standard in water), while toxicological and physiological effects have been observed with concentrations as low as 0.05-0.1 pg/mL [103], which limits its suitability as a regulatory analysis method. 

This technique is critically dependent on the quality of the mirrors and cavity, respectively. Super polishing to <0.05 nm root-mean-square of surface roughness is essential. Under such conditions the arrangement with Pellin—Broca prism exhibits a base ring-down time of about 1 ps and yields a minimum detectable absorbance change of about 32 ppm. A ring-down time of about 800 ns is found for a 1 cm square mini-cavity. 

Hence, 10 ions simultaneously absorbing power in the detector region would be expected to give a signal-to-noise ratio of 1/1. Depending upon the drift velocity, this corresponds to a minimum detectable ion current of about 10 A. 

The minimum still detectable concentration Ni of absorbing molecules is determined by the absorption path length L, the absorption cross section oik, and the minimum detectable relative intensity change A///o caused by absorption. 

In order to reach a high detection sensitivity for absorbing molecules, Loik should be large and the minimum detectable value of AI/Iq as small as possible. 

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