CA2707035A1 - High throughput methods for analysis of contamination in environmental samples - Google Patents

Abstract

Use of high throughput methods to analyze samples for toxic elements originating from industrial hygiene and environmental sampling are described. These methods utilize optical detection methods using plates with arrays and microwells Methods to fabricate samples in such plates are described The invention is particularly illustrated by demonstrating its applicability for analysis of beryllium by fluorescence This invention also discloses the use of improved filtration method compatible with the high throughput methods of sample preparation and analysis

Description

High throughput methods for analysis of contamination in environmental samples Inventor: Anoop Agrawal, et al.

Related Application/Claim of Priority This application is related to and claims priority to provisional application 61/008,229 filed on 12/19/07, which provisional application is incorporated by reference herein.
Government Rights This invention was made with US Government support under contract DE-FG02-06ER84587 awarded by the Department of Energy. The Government has certain rights in this invention.

FIELD OF THE INVENTION

[0001] The present invention relates to the detection of contaminants in environmental and industrial hygiene samples using high throughput methods. This method can be used to analyze environmental samples of soil, air, water, surfaces and any others for contamination by metals and compounds.

BACKGROUND OF THE INVENTION

[0002] Environmental and industrial hygiene samples originate from a number of places, such as industrial sites, waste storage and dumps, around these areas in air, water and soil, or those that may have been contaminated by terrorist, military or other acts.
Some of the toxic industrial materials are lead, hexavalent chromium, cadmium, mercury and beryllium to name a few prominent ones. These materials are typically analyzed by extracting the toxin or the contaminant in a liquid medium (using acids, bases and other solvents and solutions) and then subjecting this to analysis.
Typical analysis involves taking these samples and analyzing them sequentially through chromatography (e.g., high performance liquid or gas chromatography), inductively coupled plasma along with atomic emission or a mass spectrometer (ICP-AES and ICP-MS respectively). The samples are eluted into the equipment in a sequence with enough gaps or purges so that there is no cross-contamination. To decrease the labor content and increase the efficiency of the analysis, autosamplers have been developed for such instruments. In these the samples are put in a queue, and the samples are automatically analyzed one after the other. As an example in modern ICP-MS
instruments 200 samples may be queued which may take 10 hours to analyze. This causes many issues related to the drift in baseline, and for proper quantification one may require calibration standards to be run periodically during this long analysis time.

[0003] The rapid techniques developed in biological analysis lend themselves to high throughput analysis. In these methods the high throughput is obtained in two ways, first by automating the sample preparation and secondly by developing instrumentation that can analyze a large number of samples within minutes. As an example, microarray and microwell formats are routinely used and are then analyzed by optical scanners (by looking for fluorescence, luminescence and absorption/transmission changes and quantifying these). Typical microwell formats have 24, 96, 384 or 1536 or more wells in an area of about 8cm x 13cm. Such plates can be read by the optical scanners in a matter of minutes. Microarrays may have thousands of analytical spots on a plate. Further, standards occupy some of the spots or wells so that they are all read almost simultaneously (within minutes) avoiding temporal drift.

[0004] In addition to be able to read the samples rapidly, it is highly preferable to automate the sample preparation procedures which require repetitive steps of mixing various liquids, filtration, pipetting, and weighing. The purpose of this invention is to enable high throughput analysis of environmental samples. This will reduce cost and enable one to take more samples in order to ensure that safety is not compromised due to the throughput issues.

[0005] One object of the present invention is to demonstrate that environmental and industrial hygiene samples can be measured at high throughputs.

[0006] Another objective of this invention is to enable processes so that environmental and industrial hygiene samples could be prepared with high degree of automation such that they are ready for analysis.

[0007] Yet another objective is to automate the sample preparation and analysis so that high throughputs are achieved.

SUMMARY OF THE INVENTION

[0008] In accordance with the purposes of the present invention, as embodied and broadly described herein, the present invention provides a method of preparation of samples and their analysis at high throughputs. This reduces cost, increases efficiency and also reduces chemical waste generated during analysis. This invention is particularly applicable for environmental and industrial hygiene analysis (typically soil, water, air and surface) to analyze toxic elements such as lead, mercury, cadmium, arsenic, beryllium, thallium, antimony, uranium and selenium and other suitable toxic materials.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1: Schematics of a 96 well plate array;

[0010] Figure 2: Schematics of automation for sample preparation for beryllium analysis by fluorescence;

[0011] Figure 3: Schematics of filtration step in automation;

[0012] Figure 4: Change in fluorescence emission for different concentrations of beryllium when excited by optical radiation of 385nm and a bandwidth of 20nm (i.e., IOnm centered around 385nm);

[0013] Figure 5: Excitation spectrum for the peak at 476nm and at 545nm for beryllium assay, the ratio of the two spectra is also provided;

[0014] Figure 6: Emission spectra of a beryllium assay when the sample is excited by 385nm and by 390nm optical radiation, the band width for both is lOnm.

DETAILED DESCRIPTION

[0015] The efficacy of the invention will be primarily demonstrated for analyzing beryllium, but the scope of this invention is applicable to a number of environmental toxins.

[0016] Beryllium is a metal that is used in a wide variety of industries including electronics, aerospace, defense, and the Department of Energy (DOE) complexes. Exposure to beryllium containing particles can lead to a lung disease called Chronic Beryllium Disease (CBD). Recent new regulations from DOE dictate a permissible exposure limit of 0.2.ig/m3 in air, a housekeeping level of 3.ig/100cm2 on a surface, and a release level for materials after beryllium exposure where the surface contamination due to beryllium must not exceed 0.2.ig/100cm2.

[0017] Currently, thousands of surface wipes and air filters are analyzed annually for beryllium. In addition Occupational Safety & Health Administration (OSHA) has detected airborne levels of beryllium at numerous sites within the United States. In addition, at some of the sites where past beryllium activity or disposal has taken place, beryllium needs to be cleaned from the soil, down to a level of 131 mg of beryllium in each kg of soil. The popular method for detecting beryllium on a surface involves wiping an area with a filter paper, performing a microwave digestion with acid to dissolute beryllium or its compounds, and then analyzing by inductively coupled plasma (ICP) atomic emission spectroscopy (AES). For analyzing airborne samples, one draws a known quantity of air through a filtering medium and then the filter is treated in a similar fashion to the surface wipes. The ICP-AES technique also requires highly trained operators and the entire sample (typically 5 to 15ml of solution) is consumed in order to meet the detection levels. If a sample is identified as positive for beryllium then it is difficult to verify with a second run, as most or the entire sample is consumed in the first run. For air filtering one typically analyzes a filter after an eight hour shift. However, in order to protect workers from large instantaneous release of beryllium, the sampling frequency has to be increased which places a greater burden on laboratories using traditional methods. This also affects the sampling frequency for the wipes, where one has to use complex statistics to estimate the thoroughness of the sampling. Such bottlenecks can also be reduced by using the high throughput methods of this invention, where a larger number of samples are analyzed in order to improve confidence in sampling.

[0018] Optical analysis methods such as fluorescence, luminescence and absorption (or change in transmission) have been highly developed for high throughput analysis of biological samples. The fluorescent method for beryllium is well described in US
patent 7,129,093 and US patent application 2005/0221498 and PCT application W02008/130,737. All of these are incorporated herein by reference. Electron or x-ray induced fluorescence may also be used in an array format. The samples are typically made by putting probes on surfaces or liquid samples in plates with microwells.
Examples of microarrays can be found in US patents 5,700,637; 5,744,305; and 7,195,872 and US patent application 2003/0027129. Microwells have been used for a long time in biological analysis. Typical plates with microwells are available in standard wells of 24, 96, 384 and 1536 (Fisher Scientific, Pittsburgh, PA) where a typical plate size is about 8xl3cm. Samples in array or microwells can provide a high throughput analysis if the test can be configured to take advantage of this in the field of analytical chemistry. Using an autosampler on an ICP instrument can take almost 6 hours to analyze 90 samples by either AES or MS (Mass Spectrometer), or even using atomic absorption spectrometer (AAS). During this period the calibration curves may shift and one may have to check these periodically extending the analysis time further.
As a comparison, a plate with 96 wells (or samples) in a fluorescent system can be read in the order of a few minutes (usually less than 10 minutes, typically less than 1 minute). It is also preferred that some of the wells (typically 4 to 12) are occupied by the standards so that the standards are read at about the same time as the samples, and the unknown concentrations in the samples are detected by calibrating against the standards. The wells holding the calibration standards can be in a particular row or column or be distributed in any order within the plate. This is also different from conventional instruments, where the instrument has to be calibrated first in order to read the samples. In this case all the wells are read for fluorescent intensity, and then the software tool picks out the calibration wells as indicated by the user, fits a curve through it and provides the concentration for the unknown samples. Figure 1 shows a schematic of a 96 well plate. The rows and columns are designated by a matrix of letters and numbers. For example, well B4 will be the well in the second row of the fourth column. As an example standards can be in a column from Al to H1 or in to H6, or in a row or in wells distributed throughout the plate. Some of these may be standards for calibration, while the others may be standards to check or verify the accuracy of the results, particularly if some of the results are extrapolated.
It is best to use standards in the range of highest interest, and then use some of the wells with predetermined concentrations that are extrapolated and are only of cursory interest. As an example for beryllium since the regulations call for testing from 0.2 to 3 g, one may use most standards about in this range to get good accuracy. However, to test the accuracy of detection capability or to test if the values are exceeding the highest numbers by a significant amount one may use samples that are 2 to 10 times in excess or less than the highest and the lowest numbers respectively. For example one may use standards corresponding to 5, 1, 0.2, 0.05 and 0 g for calibrating the range of interest. The samples corresponding to 20 g and 0.01 g are also included to check the extrapolation outside the range of highest interest and to check the detection limit of the method respectively. Another way for large dynamic ranges is to calibrate on a log-log scale. Typically this is useful when the range of interest is more than two orders of magnitude (Le., a difference of 100 times or more). A significant advantage of the optical method is the speed at which the plates or arrays can be read.
This allows a laboratory to purchase a single machine which can process thousands of samples that replaces a bank of ICP machines which are highly expensive.
Further, in the plate readers one can typically read in a number of formats, i.e. at different wavelengths or different modes such as fluorescence, absorbance, polarized fluorescence, etc. This can be used to provide additional diagnostic tools to eliminate false signals. The false signals can be particularly strong when one is looking at ultralow concentrations, which are typically below lppb (parts per billion) in analytical solutions. As an example, in beryllium analysis by fluorescence the measurement solutions (sample solution mixed with dye solution) can turn yellow.
Thus it is important to look at the samples in absorbance/transmittance mode to separate those samples that are yellow in color. The sample yellowness can be typically seen by looking at absorbance anywhere in the range of 400 to 450nm.
Thus an optical filter transmitting in this range can be used to check this. To accurately measure beryllium in such samples, one protocol is to wait for a period of 30 minutes to 6 hours so that the yellow causing compounds precipitate and a re-filtration removes this and the beryllium can be measured. Alternatively, filtering these through hydrophilic filters (e.g. polyether sulfone and hydrophilic polypropylene) without waiting has also shown to be effective.

[0019] Further, another serious drawback of conventional methods is the labor involved in sample preparation which adds both to the cost and time. Typically samples are brought to the analytical laboratories in bulk form or as air filters or wipes which are then processed so that the analyte is extracted into a liquid medium. This preparation is usually cumbersome. This may also result in errors and fatigue leading to injury, e.g. Carpal Tunnel syndrome due to repetitive actions such as pipetting. This step can also be automated particularly for preparing arrays or microwell plates. These instruments are available for biological analysis and have not been used advantageously by the analytical, particularly the environmental and industrial hygiene industry.

[0020] As a further advantage, the sample requirement of optical methods is small, thus a small fraction of the sample is used for analysis and the rest can be stored to re-check if necessary. As an example, most ICP methods may consume 5m1 to 15m1 of sample where as for optical methods less than 2m1 is required and in many cases less than lml. The 96 well plate readers typically use less than 0.3ml per well and 384 well plate readers use less than 0.05ml per well. This also allows one to put several replicates of samples on the same plate to get high statistical accuracy. As an example, in NIOSH (National Institute of Occupational Safety and Health) methods 7704 and 9110, ASTM (American Society of Testing Materials) D7202 methods for beryllium, in general plastic cuvettes are used in which 100 l of sample and 1.9 ml of dye solution is used. These are small volumes compared to the typical ICP
based analytical methods, but these can be reduced further in well format. Smaller volume leads to low amounts of chemical usage and subsequently lower amount of hazardous waste generation as a result of conducting such tests.

[0021] One of the several reasons for not automating the sample preparation for typical analytical chemistry methods is the high volumes of liquids that are used for sample preparation even if smaller samples are used in the final analysis, such as in the beryllium fluorescence method described above. This arises as the sample in the form of a wipe or a filter or bulk soil, one needs larger volumes of these liquids to extract the analyte into the liquid medium. Typically in high throughput methods used in the biological industry, most ingredients are pipetted in 1 ml or lower quantities. In analytical chemistry one requires higher volumes and it is usual to use 5ml to 100ml liquids per sample. Another related reason for the lack of automation in the analytical chemistry field is the type of liquids used. In biological assays the liquids used are close to neutral pH (e.g., from about 4 to 9), whereas in analytical chemistry of environmental samples one typically uses strong acids and strong bases with pH
usually lower than 2 or pH higher than 10. This becomes difficult to handle in large volumes with system pumps that are generally provided, as these use metals and glass components which can corrode. Use of small disposable pipettes, which generally use dispensable polymeric tips (e.g., polypropylene), are fine with the extreme pH
range, but require several operations to dispense large volumes and reduce the throughput of the instrument in terms of samples prepared in a given time. The reason for using smaller pipettes is related to adjusting the spacing between the probes to separation of wells in the standard plates.

[0022] Some of the instruments for automated sample preparation are available from Hamilton Inc (Reno, NV) as Microstar, model 4200, 4000); from Perkin Elmer (Waltham, MA) as Janus; from Tecan Systems Inc (San Jose, CA) as Freedom EVO, Genesis; from Velocity 11 (Menlo Park, CA) as Bravo, Vprep; from Beckman (Fullerton, CA) as Biomek; and from Gilson (Middleton, WI) GX and Quad series.
All of these have multipurpose robotic functions which may include two or more functions such as picking up and dispensing liquids, filter, mix reagents, weighing, move parts from one place to the other and close or open containers, etc.. For high throughput it is better to pump the large volume fluids through the system, so that these can be added to the processing tubes quickly and in one operation. When this is done, these fluids can interact strongly with the materials of construction used. As an example to pump these fluids accurately, glass syringes and metal probes (usually stainless steel) are routinely used. However, in analytical chemistry the use of such materials with strong bases or acids could present problems as most strong acids will attack and corrode stainless steel, and hydrofluoric acid used in many digestions will etch glass. In some cases if the elemental toxin being analyzed is present in small concentration in these probes or syringes (e.g., beryllium in steel and lead in glass), then the results at finer limits may be compromised. Thus it is preferred to replace these with polymeric materials or coat them with polymers to reduce their interaction with the fluids, or use those systems that do not use syringes such as Gilson's GX 281 and GX 271. Preferably the syringes and probes (fine tubes for aspirating in liquids and dispensing them from one place to the other) should be made or coated with organic polymers. Some of the preferred polymers to make these components are polyolefins (e.g., polypropylene), halogenated polymers (e.g., polytetrafluoroethylene and fluorinated ethylene/propylene and polyvinylidene fluoride and polyvinylidene chloride), polycarbonate, polysulphone, polyacetal and polyesters (e.g., polyethylene terephthalate and ethylene naphthalate), and also thermoset polymers such as epoxies and alkyd resins. These polymeric materials along with parylene may also be used for coating metallic or glass/ceramic parts. If coated, these coatings should be placed both on the exterior and the interior surfaces of the probe, while for syringes only the interior surfaces are sufficient. In most preferred cases the probes and the syringes are constructed out of the polymeric materials, with exemplary materials listed above. In some cases it may be desirable to separate steps that require high volume of fluids that are dispensed from those which then take processed samples and prepare small quantities of analyte for final analysis. As an example, ASTM method D7458 is for bulk soil analysis. In this method 50ml of ammonium bifluoride (ABF) solution is dispensed for each sample and then this is processed by heating and after that only a fraction of a ml of the solution is needed to prepare the final analyte for analysis.
Since these vials are large and several liters of ABF solution will be required, it is desirable to preserve space on the expensive multi-purpose robotic system and to speed the analysis, a separate simple robotic system is used where the liquid is pumped through for initial pumping of ABF. After processing these vials may be placed on the multipurpose robotic system for preparation of the analyte which requires filtration, mixing with other reagents and preparation of the plate with samples and standards. In case the space on the multipurpose robotic assembly is restricted, one may design a carousel that can feed one or more vials at a time so that the fluid from these can be picked up by the multipurpose robotic probes.

[0023] The fluid handling systems may be optionally integrated with liquid level sensors, bar code readers, etc. in order to reduce manual checking and data entry. One may also include a station for automatically weighing the individual samples, where the samples (prior to positioning them in the plates) may be placed robotically.
For handling large numbers of samples it is also preferred to use cappers and decappers to easily tighten the caps and remove them from a multitude of bottles or vials used to process the samples. In the fluid handling systems water is typically used as "system fluid" and for washing the reagents as it runs through the system. The "system fluid"
is typically degassed before use so that air bubbles in the line do not cause loss of precision and reliability in dispensing which can interfere with the results.
For high sensitivity analytical analysis it is preferred that inline degassers be added at the point of entry of system fluid, or in line with each of the fluid channels. For example in line degassers are available from Phenomenex (Torrance, CA) under the brand name of Degassex.

[0024] Some of the standard methods of use in the industry to analyze toxic materials for the environmental and industrial hygiene applications are given in the table below. Most of these methods use ICP-AES or ICP-MS for analysis.

Table 1 Material to be analyzed Standard methods using ICP-AES Standard Methods using and ICP-MS, AA optical and X-ray fluorescence Arsenic OSHA ID105 EPA SW846- 6010, 6020, 7061, 7062, Beryllium NIOSH 7300, 7102, 7301, 7303, 9102 NIOSH 7704, 9110 OSHA ID125g, ID206 ASTM D7202 EPA SW846- 6010, 6020 Cadmium NIOSH 7300, 7048, OSHA ID121, ID125g, ID206, ID 289 EPA SW846- 6010, 6020 Chromium (Hexavalent) NIOSH 7605, 7604, 7600, 9101 NIOSH 7703 OSHA ID215, W4001 EPA SW846-7196 EPA SW846- 7195, 7197,7198,7199 Lead NIOSH methods 7082, 7103, 7300, NIOSH Methods 7505, 7701, 9100 and 9105 7700, 7702xrf OSHA ID121, ID 125g, ID206 EPA SW846- 6010, 6020 Mercury NIOSH 6009, OSHA ID140, ID145 [0025] The present invention is concerned with preparation and analysis of arrays of samples for environmental analysis, which are prepared using automation and read quantitatively by optical methods or by ionizing radiation such as x-rays and electron beams. Typical regulation limits for these materials are summarized in Table 2.

Table 2 Material OSHA NIOSH ACGIH EPA DOE
Arsenic Air Water g/1 10 g/m3 Beryllium (air) 2 g/m3 25 g/m3(Pea 0.5 g/m3 2 g/m3 0.2 g/m3(action k) limit) 5 g/m3(Ceili ng) Beryllium Water Surface 0.004 3 g/100 cm2 mg/1 0.2 g/100 cm2 (Release level) Cadmium Air Air Water 5 g/m3 10 g/m3 (total) 0.005mg/l 2 g/m3 (respirable) Chromium Air Air Air (Hexavalent) 100 g/m3 100 g/M3 100 g/m3 Mercury Air Air Air Water (inorganic) 100 g/m3 50 g/m3 50 g/m3 0.002mg/l Lead Air Air Air Water 50 g/m3 50 g/m3 50 g/m3 0.015mg/l ACGIH: American conference of Government Industrial Hygienists' EPA: Environmental Protection Agency [0026] As an example, for beryllium the federal regulations for the Department of Energy (I OCFR850) state that airborne contamination in the work space must be less than 0.2 g/m3, which is generally measured by personal samplers (carried by workers in beryllium contaminated area) over an eight hour shift. This is a time weighted average (TWA), where the air is sampled over an eight hour shift and the filter from the sample is then analyzed. Similar standards are established for the other toxins in the work place, particularly for lead, mercury, cadmium and others as listed in Table 1. For example, U. S. Environmental Protection Agency (EPA) standards for water contamination on antimony, selenium and thallium, where the maximum is limited to 6 g, 50 g and 2 g in one liter respectively [0027] As a first step for most methods, the contaminant is drawn from a solid matrix in a liquid solution (unless the contaminant is already in liquid, such as water).
This is done either by dissolution (or extraction of the contaminant or components including the contaminant) or by dissolving of the solid. One may use solutions from known methods to totally digest the sample in order to get the analyte in the solution. For example, for beryllium, the methods from Environmental Protection Agency (EPA) such as SW846-3051 and 3050, or OSHA125G or NIOSH 7300 use concentrated acid, such as nitric acid, which may be mixed with hydrogen peroxide and concentrated hydrochloric acid, or one may use ammonium bifluoride aqueous solution, as given in NIOSH procedures 7704 and 9110 or ASTM D7202 and D7458.

[0028] Although the above acids may be used with this invention, it is surprising that ABF
solutions are not used more often in dissolution of other toxins. This is because ammonium bifluoride (ABF) was quite effective in extracting beryllium from metals, oxides and silicates, and secondly it is a one step dissolution process. As an example, NIOSH method 7300 for beryllium calls for treating the samples with a mixture of perchloric and nitric acid on a hot plate at 120C. More of this acid is added after a small volume is left, and this is repeated several times until the solution is clear. This is then washed with distilled water and heated to dryness at 150C, and more acid solution is added to dilute the material to a specific amount and then used.
This is a multistep process whereas in the ABF treatment, the sample is taken in a tube and a predetermined amount of ABF is added. The solution is then either agitated or heated and at the end of it is filtered for further analysis. This is a single step procedure where several steps of decision making and adding of reagents are not needed to get the analyte in solution. As discussed later, any procedure may be automated;
however, one or two step methods are easy to automate at prices that most environmental laboratories can afford. Although ABF aqueous solution has been principally used for beryllium, it may also be used for extracting elemental toxins, such as antimony, lead, thallium, mercury, arsenic, cadmium, selenium, uranium and hexavalent chromium from the media (filter or a wipe) or soils. For each type of sample (air, soil, wipe or the nature of contaminant), the time of treatment, concentration of ABF, temperature of treatment and the ratio of ABF to the sample may vary. However, common protocols are always preferred for automation so that the costs can be reduced.
Typically ABF concentrations less than 20% in the solution and a temperature of less than 1000 are adequate for such extractions. For dissolution, the ratio of soil (or the amount of material on the media) to ABF in the solution is preferably less than 1. For example, it has been found that high fired beryllium oxide found in air or that deposits on surfaces in beryllium processing facilities can be dissoluted in 1 %ABF at 80C in half an hour in 5m1 solution. Beryllium metal can be dissoluted at room temperature under similar conditions in 30 minutes. When beryllium has to be analyzed in soil samples then for 0.5g of soil sample 3%ABF solution is required at 90C for 40 hours, and 50m1 of solution is required for half gram of soil samples with particle sizes less than 100 microns (Agrawal, A. et al, Environmental Science and Technology and ASTM test method D 7458). However, in most cases the solvents to extract the toxic metals are acidic in nature (pH is typically less than 4). Since the toxicity of ABF is lower as compared to the concoction of concentrated acids that are used to typically dissolute toxins for environmental and industrial hygiene applications, the use of ABF
solutions is desirable.

[0029] Multistep dissolution processes may also be automated, where in the dissolution step various optical end point checking techniques may be incorporated. For example, simple absorption or transmittance measurement optics (for example an LED with a detector may be combined) to check the dissolution tubes. The tubes may be located in an oven or a hotblock. The tubes may be capped and these may be decapped and capped automatically in order to add the reagents. Such cappers and decappers are available from FluidX (Cheshire, United Kingdom) and Par Systems (Shoreview, MN) and J-KEM Scientific, Inc (Saint Louis, MO).

[0030] Automated liquid handling systems increase speed, provide consistency in sample preparation and lower cost by reducing the labor. In addition for analysis of radioactive materials such as uranium and thorium it can also provide sample preparation without human intervention to increase the safety.

[0031] Hexavalent chromium in NIOSH method 7600 is conducted by dissolution of the chromium from an air filter in an acidic or a basic medium where one uses sulfuric acid to extract soluble chromates and sodium hydroxide and a sodium carbonate mixture to extract insoluble chromates particularly in the presence of reducing agents.
Once the final solutions for measurement are made by mixing with diphenylcarbazide, these have to be measured in a period of 2-40 minutes by looking at their absorption at 540nm. This can present challenges for a large number of samples. Using automated sample processing and utilizing a plate format as discussed above, the consistency of the results can be improved substantially while providing all the benefits listed above.

[0032] Lead can also be detected by optical means, e.g., NIOSH analytical method 7700.
This looks at development of red color and is a qualitative test. However, using similar principles quantitative tests have been developed such as Hach (Loveland, CO) LeadTrakTM system. Use of multiwell plates for analysis and automated sample preparation can expedite any of such test procedures.

[0033] As an example, a sequence of automation steps for beryllium using NIOSH
procedures 7704 and 9110 or ASTM D7202 involves the steps as shown in Fig 1, which involves preparation of a 96 well plate to be read in a fluorescence plate reader.

[0034] Step 1: The samples are typically provided in individual tubes which are usually capped. These samples are placed on a rack of the automated system. Preferably the format of the rack should be similar to the multiwall plate that would be made for measurement. As an example, for a 96 well plate a typical format is a matrix of 8 by 12. This rack may be removable or it may be a hot block. Standards may be included in some of the positions on the rack if they will be processed along with the standards. Alternatively, some of the positions may be left blank if standards of known concentrations will be placed in the equivalent positions of the multi-well plate.

[0035] Step 2: The samples in the tubes may be a filter paper or a wipe with the sample particles or it may be soil. If the tubes are capped they may be uncapped manually or automatically by the system. After decapping all or one at a time, ABF (dissolution) solution in a known quantity is added to all the tubes. The tubes are then capped (or capped one at a time after adding the reagent) [0036] Step 3: In case the hot block was used as a rack, the program to treat these tubes for a specified time and temperature is initiated, or the rack is temporarily removed for such processing elsewhere. After the samples are processed and cooled, the caps may be removed manually or automatically.

[0037] Step 4: The system picks up a specified amount of the fluorescent dye solution from a vial or a tank (or it is run to the probe from the system) and then picks a specified amount of the sample solution from one of the sample tubes, preferably after introducing an air gap (between the two fluids).

[0038] Step 5: The two fluids are simultaneously dispensed in a filter cartridge or in a deep well plate with filters placed at the bottom of each well. The fluids may be aspirated and de-aspirated several times for good mixing.
The tip is then washed or disposed for the next sample, i.e. steps 4 and 5 are sequentially repeated for all of the samples. If a filter cartridge is used for each individual solution then these are preferably arranged in a similar matrix as the well plates (e.g., 8x12 for 96 well plates), and if a deep well plate with filters is used then this also is preferably for a 8x12 format to go with the microwell plate.

[0039] Step 6: If the samples are in individual filter tubes, these may be pressurized so as to filter the contents in a matrix of elution tubes or an elution plate located below the tubes or the filtration cartridge (which ever is used). In the latter typically vacuum is used between the filter and the elution plate for filtration. If any of the compartments in the filtration cartridge are empty they should be filled with a fluid in the same volume as the other wells with samples. A fluid of choice in this case is water.

[0040] Step 7: After filtration, the filter tube rack or the filtration plate is removed so that the filtered fluids in the elution plate or the elution tubes is accessible to the probes. A specified quantity of filtrate is removed (typically same quantity for every sample) with disposable or a washable probe into a microwell plate. For 96 well plate this is typically between 100 to 300 l and for a 384 well plate this is between 20 to 50 l.

[0041] Step 8: Add standards as needed to those microwells in the plate that were reserved for this purpose, and the plate is inserted manually or automatically in a plate reader.

[0042] In step 8, standards can be pre-processed, i.e., one may provide a vial comprising a standard concentration which may be diluted in a serial fashion to make concentrations in a desired range. These serial dilutions are then mixed with the dye solution and then dropped on to the wells reserved for the standards in the same volumetric quantity as the samples. All this is done on the same equipment that is used for the sample processing above. The multiwall reading plate may be automatically inserted in the fluorescence reader or it may be done manually.
In many of the automation platforms for biological work, one uses disposable plastic pipette tips or reusable metal probes. In the dissolution of environmental samples, the step in which the toxin from the sample is extracted in a liquid media, acidic media or acids are generally used. This requires probes to be highly corrosion resistant.
Polymeric probes and tubings are preferred, some of the preferred materials are polyethylene, polypropylene, polytetrafluoroethylene and fluorinated ethylene-propylene. In one preferred embodiment both the probe and the attached tubing are made out of a plastic and the length of the tubing is adjusted so that the fluids that are picked up by the probe are confined to the tubing only and do not enter the syringes which may have metal or glass parts. The volume of the probe and the tubing is typically 50m1 or less.
If metal probes are used they should be preferably coated with acid resistant organic coatings, especially any surface that will come in contact with the acidic solutions.
Preferred materials are organic polymeric coatings with low susceptibility to moisture absorption. Some of these materials for coatings are polyethylene, polypropylene, polyvinylidene chloride, parylene and fluorinated polymers, such as tetrafluoroethylene, fluorinated ethylene-propylene and polyvinylidene fluoride,. The preferred coatings are typically deposited from vapor phase or from a solution (e.g., from an emulsion). For use with ammonium bifluoride, the most preferred coatings are polyvinylidene chloride and parylene C and parylene D, which are deposited from the solution or vapor phase and are able to coat both the exterior and the interior of the probes.

[0043] The preferred microwell plates for such analysis are those that have dark sides (preferably black) between the wells. This ensures that there is no optical contamination of signals from the adjacent wells. For fluorescence one may use black bottom plates or those with clear bottom. In the former, the background fluorescence from the substrates is reduced. Typically the excitation source is from the top of the open wells, and the readout is from the top for fluorescence or from the bottom for fluorescence and absorbance. Even with the clear bottoms, it is preferred that these plates have high absorbance for the optical wavelengths used for exciting the fluorescence signal. For beryllium, these plates may have UV absorbers to absorb the radiation below 400nm. Further, depending on the surface tension of the fluid and the walls, there is a possibility of a shaped meniscus formation at the top, which can focus and bend the incoming light in a fashion that may distort the signal from the well. It is preferred that the wells be either coated with materials to modify the surface tension so that the fluid being analyzed wets the walls of the wells (i.e., the contact angle between the liquid and the modified surface is less than 90 degrees, and more preferably less than 30 degrees). For example, for analyzing aqueous solutions these wells should have a hydrophilic coating, or one may add surfactants to the solutions (as long as this does not interfere with the analysis), to make the sides wetting and keep the meniscus largely horizontal.

[0044] Another important aspect of analyzing the well plates is the sensitivity of background to the particulate contaminants that may float on top of the wells giving rise to disturbance in meniscus and also adding to the fluorescent signal depending on the dirt. The automated processing of samples minimizes handling and reduces contamination probability. It is good practice to keep the plates covered when not in use, and handle them with gloves so that oils are not transported on to the plates, which may also add to fluorescence. Insects can have strong fluorescence, and one should examine the plates manually to ensure that bugs have not been trapped in the wells. The disturbance of the meniscus can also be reduced by adding surfactants to the solutions being analyzed. In addition, once the plate is inserted into the chamber, it may be agitated in order to wet the dirt and allow it to sink or minimize disturbances on the surface. One may optionally use a transparent plate (in the optical range of excitation and emission, e.g. quartz) in order to protect the microwells from the dust and other particulates.

[0045] Some of the coating methods employed for depositing hydrophilic coatings onto the plates is from vapors and liquids, including chemical vapor deposition processes assisted by plasma. These may be organic or inorganic. Some of the metal oxide coatings that provide hydrophilicity are comprised of silica and titania.
These coatings may be comprised of carbon to enhance hydrophilicity. Hydrophilicity may be also imparted by introducing nanopores (pores less than 100nm in size). One may use precursors such as tetra-orthoethylsilane, methyl triethoxy silane, and adjust the oxygen stoichiometry by introduction of oxygen and ozone (e.g., see WO/2007/021679). Similarly, titanium tetraisopropoxide may be used to deposit hydrophilic coatings of oxide of titanium. Both silicon and titanium precursors may also be mixed (e.g., see Nakamura, et al). These coatings only need to be present near the top rim of the wells where the liquid forms the meniscus in the wells, thus it is not necessary for these coatings to uniformly coat the entire depth of the well.
These coatings must be compatible with the solutions being analyzed and should not compromise the analytical aspects.

[0046] The surfactants may also be added to the solutions being analyzed.
These may be ionic (cationic or anionic) or non-ionic. These are preferably present in quantities of less than 0.1 % of the solution volume, and preferably less than 0.01 % so as to keep their interactions low. Some examples of such surfactants are Triton X100, Triton X-1 14, Triton X-405, NovecTM FC4430, NovecTM FC4432, NovecTM FC4434. The first three are available from Aldrich Chemical Co (Milwaukee, WI) and the last three from 3M (Minneapolis, MN).

[0047] A preferred dynamic range for beryllium quantification in surface wipes or air filter samples is less than 0.2 and more than 4 g on the media, and a more preferred range is less than 0.02 and more than 10 g on the media and the most preferred range is less than or from 0.005 to 20 g or more of beryllium on the media. This method has high flexibility to be tailored to any desired range. If higher amounts of beryllium are suspected that go beyond the instrument range, one always has the option to dilute the solutions or to use an optical filter to lower the excitation or the emission intensity.
For soils, a preferred range is from about 0.1 g of beryllium/g of soil to about 2000 g of beryllium/g of soil, a more preferred range being from about 1 g of beryllium/g of soil to about 200 g of beryllium/g of soil.

[0048] When samples are analyzed in an array format, it is easy to control the temperature and come to a quick equilibrium, most plate readers have a built in temperature control system in the sample compartment. Since the volume of material used in array format or higher is typically less then 300 l in each well. Since, air can circulate between the wells it takes a shorter time for the samples to reach thermal equilibrium.
Thus it is preferred to use sample volumes of less than 250 l, and more preferably less than 50 l. Such analysis also consumes less materials and generates less chemical waste as discussed above The thermal load on the samples is also reduced in an array format, as the optics scan a well for a short period of time, which is typically less than a few seconds, and in most cases a fraction of a second.

[0049] Further, a fluorometer equipped to look at absorbance and fluorescence is most suited for this method. Absorbance is used to measure the yellowness of the solution to see if the results will be compromised due to the presence of excess iron or titanium. Figure 3 shows an example of the spectra where the sample has beryllium and iron. If the samples are yellow, one can wait for a period to precipitate so that the solutions can be filtered again (usually through a filter size of less then or equal to 2 microns can be used, a preferred filter used had a pore size of 0.45 microns unless mentioned otherwise). The waiting period is typically between 30 minutes to 6 hours.
Alternatively, the measurement solutions may be filtered almost immediately (much smaller waiting times of less than 30 minutes, preferably less then 10 minutes) by filtering through a smaller pore size filter such as smaller than 0.25 microns, preferably less then 0.1 microns. Further, using hydrophilic filtration media, one can eliminate the waiting time. Some of the preferred hydrophilic media are polyether sulfone (PES), hydrophilic polypropylene, etc, In such cases, the preferred filter pore size is smaller than 0.5 m. As seen in Figure 5, the yellowness can be measured by measuring absorption or transmission in a wavelength range of 250 to 650 nm, preferably between 350 to 450nm. The same lamp that is used for excitation may also be used for measuring the absorption with a different detector. Many of the optical plate readers are able to simultaneously read the plates in several modes, e.g., fluorescence and absorbance (e.g., by using clear bottom plates), and since these are fast, they could also read at different regions of the spectrum, allowing multiple analysis to test for anomalies and provide a cross check on the data.

[0050] Filtration of multiwell plates is an important process and one needs to assure that after filtration there are no droplets hanging at the bottom of the filter plate.
Fig 3 shows a preferred setup for filtration process. The robotic arm of the automated instrument is shown as 31 that assembles and/or disassembles the filtration set-up. The filtration setup comprises of a reservoir plate (37) with wells to contain the filtrate.
On top of the reservoir plate, a housing (38) through O-rings (39) connects a filter plate (34). In some cases instead of "0" rings flat joints are also used. This is located with aligned individual filters cells, each having an individual filter (35). For filtration process, vacuum is pulled in the housing (from 30), so that the liquid passes through the filter and is collected in the reservoir plate (37). The vacuum is typically in the range of 25cm to 55cm of mercury, with a preferred range being 40 to 55cm of mercury.
At the end of this, all the components above the plate 37 are removed robotically so that the liquid probes can access the filtrate (37a) in plate 37 and continue with the analysis.
It is found that at the bottom of several of the filter plate tips a liquid droplet remains (as shown by 36). This is not acceptable, as during the removal of this plate after completing the filtering process, these drops may be released and contaminate liquid in the other wells in the reservoir plate 37. To overcome this, so that all the liquid is drained in the filtration process, we found that sealing the top of the wells in the filter plate (34) is important while the vacuum is pulled for the filtration step. In addition, sealing the wells properly may not require that all the wells in the filter plate 34 need to have fluid in them to balance out the resistance to flow in each well. In the past, membranes have been used to cover the top opening of the wells, but it does not effectively seal all the wells to the point that after filtration none of them have any droplets (36) left. There are vacuum assist devices available for microplates, such as those from Whatman (Florham Park, NJ) with a product number 7705-0112. These use a pressure sensitive adhesive sheet so that it can be placed on the filter plate and then pressed or squeezed in order to seal each of the wells at the periphery, however, this introduces a manual step in the automation where the pressure sensitive adhesive needs to be pressed and rolled several times to ensure good sealing. Further, this manual step can lead to poor sealing if creases are trapped.

[0051] This sealing problem can be accomplished by a plate with individual elastomeric plungers, or plungers with individual O-rings that can seal every well around the perimeter. However, a simple innovative way of overcoming this was by using a thin elastomeric sheet which is pressed against the wells. In order to make this most effective, it is preferred to combine two sheets of flexible materials in the following way; the thin sealing sheet (first flexible membrane) with a layer of another soft material (32c) called the cushion (second flexible membrane). The cushion helps in transmiting the pressure applied to it uniformly to the first flexible membrane (32a). A
preferred way to apply pressure is by a dead weight 32b (e.g. a plate with an appropriate mass). The cushion layer 32c is usually thicker than the element 32a, and may be made out of a flexible open cell foam, closed cell foam, gel or viscoelastic pad or an elastomer generally ranging in thickness from about 0.2cm and thicker, preferably thicker than 0.5cm. The element 32c may be bonded to the dead weight 32b. The characteristics of 32a are very important as it needs to seal each of the wells and be flexible so that it does not pucker or crease while sealing. We found good results with elastomeric sheets that were 40 durometer or less in hardness and a thickness less than lmm. More preferably durometer 20 or lower and a thickness of 0.5mm or lower. This allowed the thin elastomer to drape around the top opening of each of the wells without leakage. The element 32c transfers the force from the dead weight or another mechanism more uniformly onto 32a, but does not have to bend with the same degree of precision as element 32a. The thinness of 32a also compensates for any nicks, molding reliefs, flow welds around the well perimeters that are difficult to seal with a thick sheet. Another important parameter was the pressure on this elastomer film. This was greater than 2.5g /cm2 and preferably greater than l Og /cm2 of the total filter plate area 34 (including the cross-section of the wells) as projected normally. Some of the preferred materials of construction for 32a are silicone, polyurethane, Viton , ethylene-propylene diene monomer (EDPM) elastomers, polybutadiene, fluoroelastomers, polychloroprene and polyisoprene.
32c can be constructed from a wide variety of materials including the ones described above for 32a as a solid material or as a foam including cellulosic materials and polyolefins (specific examples being, closed cell foams made out of polyethylene, silicone, polyurethanes and ethyl vinyl acetate). The foams should be preferably soft characterized by firmness, typically less than 25% deflection when subjected to a force of less than about 10 psi (typically tested using ASTM method D5672).There may be additional members or sheets with similar or different mechanical properties and thicknesses that may be added between the elements 32b and 32a, and may be optionally bonded together and even to the dead weight to form a composite. It is preferred that the membrane 32a is not bonded to the upper members so that it can deform freely. In order to make the system user friendly, it is preferred that all members (dead weight or pressure plate along with flexible elements other than membrane 32a) are bonded together. Since the bonding assembly will be used several times it is important that all the bonded flexible materials have a high resiliency.
Membrane 32a may be replaced after each filtration step or it may be reused.
In addition, it may be necessary to slightly restrain the edges of the first membrane (32a) so that this membrane is not pulled in due to the suction created in the wells by the vacuum process in filtration. One method may be too make the membrane 32a long enough so that it wraps around all the preceding elements including the dead weight and then held by removable mechanical clips, magnetic strips or pins so that it is easy to assemble for robotic operations by taking these on and off. The deadweight may be made of magnetic material, or magnets or magnetic strips may be attached to it to which the removable magnetic strips/pins can be assembled to. The sealing of the well tops in this invention is particularly good and can be seen when filtering those well plates, where the liquid levels in them is different, or in some wells no liquid is present. This method ensures that the filtration process proceeds smoothly even under these circumstances. In some cases where a good sealing of the wells is not required due to the construction of the multi-well plates, liquids used or otherwise, one may do away with the element 32a, however the use of this membrane results in very high confidence in the filtration process performance.

[0052] For the beryllium method using fluorescence the sensitivity can be increased by changing the ratio of the sample containing dissolution solution and the dye solution.
In US patent 7,129,093, the volumetric ratio of the dissolution solution (comprising beryllium) to the detection solution (comprising dye) was 1:19. We found that ratios higher than 1:19 may be used to increase the detection limit of the method while keeping the other parameters constant. Increased ratios result in more beryllium in the detection solution thus increasing the sensitivity (lowering the detection of beryllium on the original media) of the method. Ratios higher than 1:12, e.g.
such as 1:4 may be used to increase the beryllium content in the "measurement solution" by four times. Use of dilution modification to increase sensitivity has been published by Ashley et al, in Analytica Chimica Acta in 2007. The automation for any dilution (e.g.
20X or 5X or a number in between) can be easily achieved by change of a software protocol. Several methods can be provided for the user to select. Since only a small quantity of the sample is used, both protocols can be run automatically, or sequenced so that if no beryllium is detected (or detected below a certain level) a separate assay is run for these using a 5X dilution.

[0053] For beryllium analysis using HBQS dye, a preferred excitation a range is between 365 to 395nm. This maximizes the emission signal response between 470 and 480nm and maintains good linearity. For filter based readers an excitation filter transmitting at 385nm with a bandpass of equal or less than 20nm, preferably less than or equal to I Onm results in high excitation. For emission, the peak transmission in the range of 470 to 480 with a bandpass of lOnm is preferred. For example, a preferred filter will have a peak transmission at 475 nm with a bandpass of 5nm. The most preferred filter will have a plateau in the peak transmission area, with the transmission dropping to about 0% in about 2nm on either side of the curve. The transmission at the peak should be preferably greater than 50%.

[0054] Another, factor that leads to improvement in ultra-low detection is reducing the background fluorescence. Background fluorescence from the solution may be substantially reduced by using higher purity materials, such as lysine, that have low fluorescence.

[0055] In NIOSH method 7703, hexavalent chromium is analyzed using absorption at 540nm. For this test clear bottom plates are used for analyzing these on a plate reader.
In sample preparation step centrifugation can be easily accommodated in the automation for liquid handling as in the biological systems from Tecan (Durham, NC), Velocity 11 (Menlo Park, CA), Perkin Elmer (Waltham, MA) and others.
Further after diphenylcarbazide is added there is a window of about two to forty minutes in which the sample must be analyzed. With manual methods it becomes difficult to control this time. In automated system, the addition of this material to the chromium comprising samples can be accomplished in minutes and transferred to an automated shaker (several plate readers have built in shakers as well e.g., Biotek's (Winooski, VT)Synergy 2 instrument), and then all the samples analyzed within minutes, thus increasing the precision of the measurements. For hexavalent chromium, typical limits established by various agencies in air are from 0.1 to 0.001 mg/m3 .

[0056] Example 1: Treatment of Stainless steel probes for increased corrosion resistance.

[0057] Stainless steel needles and discs were exposed to 3 weight % ammonium bifluoride solution in water at both 25 and 90 C. The stainless needles were unprotected whereas the discs were coated with a protective material by Restek Performance Coatings (Pittsburgh, PA) namely Silosteel-CR. Silosteel-CR is a corrosion resistive layer that increases the lifetime of materials in acidic environments. The two forms of stainless steel were soaked in the ammonium bifluoride and their weight monitored with time.
The results are summarized in Table 3. As seen in the table all samples did not do well and were corroded by ammonium bifluoride. At 90 C the sample with Silosteel-CR layer lost 4 weight % and the soaking solutions turned green in color.
Table 3:
Sample Soaking Time Initial Final A % Comments Material Temp. left Weight Weight Weight Weight ( C) soaking (g) (g) (g) loss (hrs) Stainless RT 112 0.11626 0.11315 0.00311 2.7 Needle is steel corroded, needle ABF
solution is colorless Stainless 90 112 0.11440 0.07621 0.03819 33.4 Needle is steel corroded, needle ABF
solution is green Stainless RT 24 7.78184 7.77656 0.00528 0.07% Disc is steel disc unchanged With CR
coating Stainless 90 24 7.76427 7.46242 0.30000 4.0% Disc is steel disc corroded, with CR ABF
coating solution is green [0058] Example 2:

[0059] Stainless Steel coupons were coated with parylene using a vapor deposition process.
The type of parlyene used was "C" and the coating thickness was 2.5 microns.
The coatings were deposited to MIL-1-46058C specifications by Advanced Coating (Rancho Cucamonga, CA). The coupons were tested in 3 weight % ammonium bifluoride at 25 and 90 C and after 48 hours showed a slight increase in there weight due to water uptake of the polymer coating. The results of the test are shown in Table 4 and show that after 48 hours the stainless steel coupons were completely protected from corrosion by the polymer coating. In all cases the soaking solution remained colorless.

Table 4:
Sample # Initial Weight 48 hour Soak (g) Final Weight (g) 25 C Soak K442A 0.19793 0.19797 K442B 0.2094 0.20945 J887 0.19408 0.19413 K462 0.40903 0.40905 90 C Soak K052B 0.1634 0.16368 K442B 0.44011 0.4699 J887 0.18734 0.1909 [0060] Example 3:

[0061] The NIOSH method 7704 and 9110 using fluorescence for detecting beryllium was adapted for a fluorescence reader using a 96 well plate. The reader was a BioTek Synergy 2 fluorescence plate reader and the plate was a Corning Costar 3915 flat bottom non-treated non-sterile, black, polystyrene 96 well assay plate. The plate format was such that the 8 wells in the first column contained the beryllium standards and the remaining wells contained the unknowns. The excitation filter used was 365 10nm and the emission filter was 476 3.5nm. The light source was a tungsten lamp and readings were taken from the top of the well (50 readings/well) with a mirror optics position of 400nm.

[0062] Beryllium Standards Preparation [0063] The standards used to calibrate the BioTek reader were 0, 0.1, 0.5, 2.0, 10.0, 40, 100.0 and 200.0 ppb and the volume in the well was 230 L. These were plotted in a linear form and a regression fit was used to calculate the correlation value R.
Values of R >_ 0.999 were considered a good fit. The 0, 0.5 2.0 10.0 and 40.0 ppb calibrants were prepared by a 20X dilution of the following beryllium standards: 0, 10, 40, 200, 800ppb supplied by Spex CertiPrep Metuchen, New Jersey. An example of the calibrant preparation is as follows: 0. lml of the Spex standard was dissolved in 1.9 ml of the dye detection solution (20X dilution) and 230 L of this solution was placed in the well. Some of the other standards from SPEX were diluted with I% ABF and then diluted 20X with the dye solution to obtain calibrants with 0.05, 0.1 and 200pp of beryllium. In these standards the source of beryllium was beryllium acetate.

[0064] Analysis [0065] The samples in the plate were solutions containing known beryllium acetate concentrations in the range 0.05 to 200ppb. The complete 96 well plate was read in a dual format where samples with beryllium <_ 40ppb were read using a "Fine"
standard calibration curve based on 0, 0.1, 0.5, 2.0, 10.0 and 40ppb standards and samples with "Coarse" beryllium content (>_ 40pb) where read using 40, 100 and 200ppb standards.
This was achieved by programming the reader to read the well plate using the "Fine"
and the "Coarse" standard calibration curves. For the "Fine" calibration reading the voltage was set at 145 volts which was equivalent to 1.76 million counts for the 40ppb standard. For the "Coarse" readings the voltage was set at 120V, which is equivalent to 1.89 million counts for the 200ppb well. At 50 reads per well and performing the dual measurement, it took 8 minutes to read the complete plate with 96 wells.
The results for the "Fine" and "Coarse" measurements are shown in Table 5.
According to the NIOSH procedures, 0.05ppb corresponds to 0.005 g on the air filter or the wipe and 200ppb corresponds to 20 g.

[0066] Table 5: Readings from a plate comprising of standards and samples A 0(0) 0.072 0.055 0.06 0.124 0.142 0.137 2.021 2.073 2.049 203.212 204.053 B 0.09 0.061 0.057 0.058 0.108 0.102 0.108 2.08 2.004 2.009 204.972 203.76 C 005 0.056 0.073 0.056 0.111 0.256 0.119 2.01 1.991 1.977 200.733 203.718 D 1.895 5 0.053 0.06 0.056 0.102 0.115 0.102 1.97 2.016 1.997 202.801 200.155 E .94 0.055 0.053 0.058 0.108 0.103 0.104 1.984 1.989 1.96 200.698 200.63 F 3(40)9 0.052 0.049 0.067 0.107 0.107 0.108 1.972 1.893 1.949 >208.000 198.995 G 1005 0.046 0.043 0.06 0.223 0.106 0.106 1.954 2.099 1.951 206.617 203.464 H 199. 0.053 0.05 0.05 0.1 0.106 0.109 1.901 1.976 1.932 204.959 200.694 (200) 1 Stds* 0.05 0.1 2 200 [0067] *Wells Al to H1 had standards that were use to calibrate and quantify the other wells.
Readings of standards against the calibration are shown [0068] The data from the above table is summarized in Table 6 where the mean and standard deviation of the values are shown. The mean for the 0.05 to 2ppb is based on readings from 24 wells and for the 200ppb sample it is based on 16 wells.
Table 6: Mean and Standard Deviation of the Unknown Beryllium Samples Sample Mean (ppb) Standard Deviation 0.05 ppb 0.056 0.007 0.1 ppb 0.121 0.038 2 ppb 1.99 0.051 200 ppb 202.631 2.184 [0069] Example 4:

[0070] A 96 well plate similar to that described in example 3 was read on the BioTek reader where the calibration curve used (standards same as in example 3) was plotted on a log-log scale. Using this scale all standards could be read in one reading which eliminated the need to do the dual scan of the low and high standards. For 50 reads/well the time to scan the plate was 4 minutes. For this reading the excitation filter was 365 5nm, the emission filter was 476 3.5nm and the sensitivity was set at 85V which is equivalent to 1.45 million counts for the 200ppb sample. The results for this analysis are summarized in Table 7 in terms of mean and standard deviation.

[0071] Table 7: Mean and standard deviation for readings based on log-log standard calibration plot Sample Number of Mean (ppb) Standard Deviation Wells 0.05 ppb 8 0.05 0.008 0.1 b 8 0.103 0.008 0.5 ppb 8 0.536 0.025 2 b 16 2.056 0.052 ppb 8 9.712 0.252 40 ppb 16 41.105 0.794 100 ppb 16 93.765 2.908 200 ppb 8 191.856 2.857 [0072] Example 5:

[0073] To modify the wetting of the samples and standards in the polystyrene wells a surfactant, namely Triton-X100, available from Sigma- Aldrich (Milwaukee, WI), was added to the solutions. The surfactant was added to the detection solution in a concentration of 0.00045 weight%. The reading was performed as described in Example 3 using both the high (sensitivity 108V) and low (sensitivity 120V) standard method. The addition of the surfactant enhanced wetting of the wells and Table shows a summary of the results for known samples at 0.1 and 100ppb.
Table 8 Sample Number of wells Mean (ppb) Standard Deviation 0.1 ppb 82 0.091 0.031 100 6 100.404 1.624 [0074] Example 6:

[0075] Solutions with various beryllium concentrations were made as in Example 3 and evaluated in 4m1 plastic cuvettes on a Shimadzu (Columbia, MD), Model RF5301PC

fluorometer. These results are shown in Figure 4 for a few of the concentrations. The emission at 476nm is used to quantify the results. This spectra was generated by using excitation at 385nm 10nm. The peak at about 550nm is due to the binding of protons.
The rationale for the choice of excitation wavelength can be seen in Figure 5.
This figure shows the intensity of the emission peaks at 476nm and 545nm as the excitation wavelength is changed. The peak at 476 sees maximum excitation from radiation at 385nm, whereas the peak at 545nm sees the maximum excitation from 370nm. Since, for quantitative aspect it is important to curb the peak at 545nm, the figure also shows the ratio of the excitation at 476vs excitation at 545nm.
This ratio peaks at 387 to 390nm. This means if this wavelength was used for excitation, one would get the highest signal at 476nm when relatively compared with the signal at 545nm. It can be seen from the excitation curves in Figure 6, one is able to curb the peak at 545nm while only loosing a bit of intensity at 476nm. Thus the most preferred excitation should be centered at 387 to 390nm, however a preferred range for the excitation peak is between 375 to 395nm Thus the range from 375 to 395 is preferred for exciting the peak at 476nm for quantitative purposes. Further it is preferred that for reading one should look at most preferably between 470 and 480nm to read the peak at 476nm, although one may consider the range of 460 to 490nm. A
preferred bandwidth for excitation is 25nm or less and for emission the preferred bandwidth is 25nm or less. These ranges may be used for both cuvette and plate readers.
These peak values and bandwidths can be used for specifying the optical filters used for this purpose. For filter based instruments, filters meeting these specifications are available from a variety of sources such as Semrock (Rochester, NY), Omega (Brattleboro, Vermont) and Barr Research Associates (Westford, Massachusetts).
A
preferred transmission of the filters is greater than 50%, and more preferably in the range of 80 to 95%. The edge should be sharp dropping from about maximum to 99%
of the minimum within 5nm.

[0076] Example 7: Filtration system [0077] An automated filtration system was implemented using the current innovation on a JanusTM automated liquid handling workstation from Perkin Elmer (Waltham, MA).

This system was used to automate the procedure for beryllium analysis by fluorescence as provided in the NIOSH and ASTM procedures for wipes and air filters. The vacuum filtration system was also provided by Perkin Elmer. The filter plate was a 96 well plate with well capacity of lml each and used 0.2 m hydrophilic polypropylene filters in these wells. This was purchased from Pall Corporation (Ann Arbor, MI) as AcroprepTM 96 Filter Plate (part number 5052). The filtrate was collected in a 96 well, 1 ml capacity plates from Corning (Coming, NY) in Costar plates with a product number of 3959. To ensure that the filter process was clean, that is each of the well openings at the top were sealed, we used a thin silicone membrane (first flexible membrane) which was a durometer hardness of Shore Al 0 and a thickness of 0.25mm. this was backed by a general purpose cellulosic sponge as "second flexible membrane" (this open cell sponge is usually used for cleaning) in a thickness of about 1.8cm (ACE brand purchased from Ace Hardware (Oak Brook, IL)) with a product number 10419, and then by placing a dead weight of 300g with an area of about 7.3cm x11.3cm. The filtration process was tested by using about 0.9ml of water in each of the wells. This process worked very well and no drops were seen hanging at the bottom of the Filter Plate after the filtration process concluded. In another experiment when the silicone sheet was replaced by another silicone sheet of the same dimensions but with a durometer hardness of Shore A20, similar results were seen. When the filter well plate was left open at the top, drops were seen hanging at many of the well tips at the bottom of the wells at the end of the filtration process. Other membranes were also evaluated as below. The same was seen when only the first flexible membrane was used which was a Viton rubber sheet in a durometer hardness of 75A. Various thicknesses (0.8mm, 1.6mm and 2.4mm) of this elastomer were tried without success. Cork sheet in a thickness of 2mm and silicone elastomer in a thickness of 1.6mm (shore A 40) as only the first flexible membrane also did not yield satisfactory results. In another experiment the silicone first membrane was backed by a soft 6mm thick closed cell polyethylene foam sheet (stiffness was about 4-6psi for 25% deformation) and then with a 6mm thick Viton sheet of durometer hardness 75A along with a dead weight of 300g made out of aluminum, all of which had a cross-section area to cover the filter plate below without hitting the edge ridges of the filter plate. This combination also worked very well with a vacuum of about 53cm mercury. In yet another experiment Viton sheet as above and the PE foam as above were purchased with adhesive back. The Viton was bonded to the steel plate and the foam sheet was bonded to the Viton . This bonded combination with a thin silicone first membrane described above also worked very well. This invention is particularly useful when filtering those well plates, where the liquid levels in them is different, or in some no liquid is present.

[0078] While this invention has been described as having preferred sequences, ranges, steps, materials, structures, features, and/or designs, it is understood that it is capable of further modifications, uses and/or adaptations of the invention following in general the principle of the invention, and including such departures from the present disclosure as those come within the known or customary practice in the art to which the invention pertains, and as may be applied to the central features hereinbefore set forth, and fall within the scope of the invention and of the limits of the appended claims.

Claims (21)

1. A method of determining the presence or amount of beryllium or a beryllium compound, wherein the detection comprises employing a solution which is fluorescent in a concentration proportional to the amount of beryllium, and the fluorescence is measured from the said solution that is dispensed in the wells of a multiwell plate.

2. The method of Claim 1, where the said solution is prepared by dissoluting beryllium particulates from air, wipe or soil and admixing this with a fluorescent dye solution.

3. The method as in claim 1 where the multiwall plate is filled with the analyte using an automated liquid handling system.

4. The method in claim 1 wherein the multiwall plate has 24, 96, 384 or 1536 wells.

5. The method in claim 4 where some of the wells in the said plate contain standards with predetermined amount of beryllium.

6. The method as in claim 1 wherein additionally optical absorption or transmittance measurements are conducted.

7. The method in claim 1 where the fluorescence signal is excited in any range that includes optical radiation from 375 and 395nm, and the fluorescence emission is measured in any range that includes optical signal between 460 and 490nm.

8. An optical method of determining the presence or amount of lead, mercury, cadmium, arsenic, beryllium, thallium, antimony, uranium and selenium, in an environmental or an industrial hygiene sample, and wherein the method comprises (a) dissolution these materials in a liquid medium unless these are in a liquid, (b) mixing this liquid medium with a material that causes an optical change in proportion to the concentration of the material to be detected, and (c) dispensing this liquid mixture in the wells of a multiwell plate for optical measurement.

9. An optical method in claim 8 where the said optical method measures at least one of absorption, transmittance reflection or fluorescence.

10. An optical method as in claim 8 where the multiwall plate is filled with the analyte using an automated liquid handling system.

11. An automated liquid handling system for preparing measurement fluids for analyzing environmental and industrial hygiene samples, wherein such system comprises of a polymeric syringe pump or a glass syringe pump with polymeric coating and a metal probe coated with a polymeric material.

12. An automated liquid handling system as in claim 11 where the polymeric coating material is one of polyvinylidene chloride, polytetrafluoroethyele and parylene.

13. An automated liquid handling system as in claim 11 where a liquid filtration step if used utilizes hydrophilic filters.

14. An optical method as in claim 8, wherein the said liquid mixture comprises of a surfactant.

15. An optical method as in claim 8, wherein the said wells of the multiwall plate are coated with a hydrophilic material.

16. An automated liquid handling system or a manual system for preparation of measurement fluids for analyzing them in a multiwell format, wherein the process of said preparation comprises a vacuum filtration step, in which the fluids in a multiwell filter plate are filtered through individual filters located in each of the wells and the said filtration process comprises of, (a) covering the opening on the top of the wells in the plate by the bottom surface of the first flexible membrane, (b) covering the top surface of the said first flexible membrane with a bottom surface of the second flexible membrane, (c) applying a pressure with an element on the top surface of the second membrane that is transmitted to the first membrane in order to seal the openings of the top of the said wells in the multiwall plate,

17. An automated liquid handling system for preparation as in claim 16 wherein the first membrane is less than 1 mm thick and has a shore hardness of less than 40A.

18. An automated liquid handling system for preparation as in claim 17, wherein the first membrane is elastomeric.

19. An automated liquid handling system for preparation as in claim 16 wherein the said pressurizing element is a dead weight.

20. An automated liquid handling system for preparation as in claim 16 where additional flexible membranes are introduced between the said pressurizing element and the first flexible membrane.

21. An automated liquid handling system for preparation as in claim 16 and claim 20 where all flexible membranes excluding the first membrane are bonded together and to the said pressurizing element.