Sample purification

Fluorescence Interference. The historical drawback to widespread use of Raman spectroscopy has been the strong fluorescence background exhibited by many materials, even those which are nominally nonfluorescent. This fluorescence often arises from an impurity in the sample, but may be intrinsic to the material being studied. Several methods have proved useflil in reducing this background. One of the simplest is sample purification. 

M. Jemal, D. Teitz, Z. Ouyang and S. Khan, Comparison of plasma sample purification by manual liquid-liquid exti action, automated 96-well liquid-liquid extraction and automated 96-well solid-phase exti action for analysis by high-perfoimance liquid cliro-matography with tandem mass spectrometiy , 7. Chromatogr. B732 501-508 (1999). 

Low resolution MS yields specificity comparable to that of high resolution MS, if a relatively pure sample is delivered to the ion source. Either high resolution GC or additional sample purification is required. To obtain sufficient specificity, it is necessary to demonstrate that the intensities of the major peaks in the mass spectrum are in the correct proportions. Usually 10 to 50 ng of sample is required to establish identity unambiguously. Use of preparative GC for purification of nitrosamines detected by the TEA ( ) is readily adaptable to any nitrosamine present in a complex mixture and requires a minimum of analytical method development when new types of samples are examined. 

Specifically for triazines in water, multi-residue methods incorporating SPE and LC/MS/MS will soon be available that are capable of measuring numerous parent compounds and all their relevant degradates (including the hydroxytriazines) in one analysis. Continued increases in liquid chromatography/atmospheric pressure ionization tandem mass spectrometry (LC/API-MS/MS) sensitivity will lead to methods requiring no aqueous sample preparation at all, and portions of water samples will be injected directly into the LC column. The use of SPE and GC or LC coupled with MS and MS/MS systems will also be applied routinely to the analysis of more complex sample matrices such as soil and crop and animal tissues. However, the analyte(s) must first be removed from the sample matrix, and additional research is needed to develop more efficient extraction procedures. Increased selectivity during extraction also simplifies the sample purification requirements prior to injection. Certainly, miniaturization of all aspects of the analysis (sample extraction, purification, and instrumentation) will continue, and some of this may involve SEE, subcritical and microwave extraction, sonication, others or even combinations of these techniques for the initial isolation of the analyte(s) from the bulk of the sample matrix. 

FTICR-MS is capable of powerful mixture analysis, due to its high mass range and ultrahigh mass resolving power. However, in many cases it is still desirable to couple a chromatographic interface to the mass spectrometer for sample purification, preconcentration, and mixture separation. In the example given above, DTMS under HRMS conditions provides the elementary composition. Apart from DTMS, PyGC-MS can be performed to preseparate the mixture of molecules and to obtain the MS spectrum of a purified unknown. Direct comparison with the pure reference compound remains the best approach to obtain final proof. 

With further work on sensor arraying, more effective means of sensor referencing may be developed to eliminate the effects not only of temperature but also nonspecific binding and test sample contamination. The overall objective is to create label-free optical sensor arrays that require minimal if any temperature stabilization, and as little a priori sample purification as possible. 

Need for High-Throughput Sample Purification and Clean-Up in Drug Discovery...2... 

NEED FOR HIGH-THROUGHPUT SAMPLE PURIFICATION AND CLEAN-UP IN DRUG DISCOVERY... 

Analysis time is typically of the order of minutes to hours depending on the sample. Normally the time spent in actual AMS analysis is not the constraining factor, but rather sample purification prior to the spectrometric analysis. Accelerator mass spectrometers are space demanding facilities that typically occupy hundreds of square meters. Normally, dedicated personnel operate the device. Considerable effort is directed into refining the methods to allow operation by smaller, less costly facilities. 

CIEF was also used to follow the production of recombinant antithrombin III (r-AT Iff) in cultures of hamster kidney cells.111 r-AT III inhibits serine proteases such as blood factors (IXa, Xa, and XIa) and thrombin. Interference by the media from which the samples were collected posed some difficulties because some of the media components have similar characteristics to those of the compounds of interest. CIEF was used to determine the pis of the separated components after sample purification by HPLC. Three major peaks showed pis of 4.7, 4.75, and 4.85, and three minor peaks had pis of 5.0, 5.1, and 5.3. These data closely resembled the data already published for serum AT III based on conventional IEF. 

Equation (32) shows that mass fractionation by simple interpolation between standards, i.e., without internal or external isotopic normalization, is only correct when mass fractionation changes smoothly upon alternating between standards and samples, which requires extremely strict sample purification. A heavy sample matrix will hkely result in Pspu falling outside the range - P j and thus nullify the basic assumption of the method. 

Sample-standard comparison is more applicable in MC-ICP-MS, in which instrument mass fractionation is fundamentally a steady state phenomenon (Marechal et al. 1999). This method has been used successfully for some non-traditional stable isotopes, particularly involving Fe, in which analyses of samples are bracketed by standards to cope with systematic instrumental drift (e.g., Zhu et al. 2002 Beard et al. 2003). However, other methods have been used for Mo stable isotope work published to date because of concerns about non-systematic changes in instrument mass fractionation, particularly arising from differences in matrices, between samples and standards. Such concerns are more acute for Mo than for Fe and many other elements because Mo is a trace constituent of most samples, increasing the challenge of rigorous, high-yield sample purification. 

The reaction scope is further enlarged to unsymmetrically disubstituted derivatives. Using one mole equivalent of two different aryllithium precursors at a time in a sequence of four micromixers and microtube reactors (MRi ), it was possible to obtain an unsymmetrically disubstituted final diarylethene. The yield was determined after solvent evaporation and sample purification on the column. 

Reacts rapidly (in less than 1 min at 20°C) with both primary and secondary amines. Forms highly stable derivatives. It does not require intensive sample purification and extraction of derivative excess. Detection limits picomoles. 

Using immobilized -glucuronidase reactors, estriol and estradiol glucuronides have been determined in urine by a column-switching technique (270, 271). Both glucuronides were hydrolyzed by the immobilized enzyme at pH 7. The steroid mixture was subsequently separated by gradient elution on a reversed-phase column, to be finally detected by UV absorbance at 280 nm. In this procedure, the activity of enzyme did not alter even after 150 h continuous run and exposure to a mobile phase containing 10% methanol. When a separate reversed-phase precolumn was inserted in the LC system, additional sample purification and shorter analysis time could be attained (272). 

Perform additional sample purification step (e.g., column chromatography0)... 

Chemical sensing applied to "as is" material requires an analytical system which is tolerant of the sample as it exists. Thus, the analyte must be determined in the matrix in which it is found. This precludes concentration steps, dilution steps, sample purification or workup of any sort and particularly makes sophisticated analytical separations prior to monitoring impractical. 

Use of internal standards. Mann and Jaworski (31) reported that when the recovery of 1-TI C IAA is monitored during a sample purification procedure, considerable loss of IAA can be detected. Bandurski and Schulze (32) suggested the use of reverse isotope dilution to help quantify the actual loss of IAA during sample analysis. In this procedure, one adds a trace amount of radio-labeled compound which ideally is identical to the compound being monitored. High specific activity is required so that statistically significant amounts of isotope can be detected without having to add an excessive quantity (mass) of internal standard. The amount of internal standard must be less than the amount of PGS. One may then accurately determine the recovery efficiency of the internal standard and thus of the PGS (32). 

Paegel, B.M., Yeung, S.H., Mathies, R.A., Microchip bioprocessor for integrated nanovolume sample purification and DNA sequencing. Anal. Chem. 2002, 74, 5092-5098. 

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