WO2000017627A1

WO2000017627A1 - Fluorescent standards

Info

Publication number: WO2000017627A1
Application number: PCT/US1998/024669
Authority: WO
Inventors: Suzanne Chin Martinez; Michael F. Mokelke; Lewis J. Throop
Original assignee: Turner Designs
Priority date: 1998-09-23
Filing date: 1998-11-19
Publication date: 2000-03-30
Also published as: AU1591299A

Abstract

An apparatus having particular use as a fluorometric standard, the apparatus including a housing adapted to be placed within a fluorometer and containing a standard material, e.g., in the form of a fluorescent fiber. The housing, in turn, provides one or more apertures configured to provide a predetermined absorption, excitation and/or emission level in the course of using the standard within the particular device. The aperture(s) can, independently, be either fixed or adjustable.

Description

FLUORESCENT STANDARDS

TECHNICAL FIELD The present invention relates to the field of fluorometry, and in particular including methods of calibrating of fiuorometric spectrophotometers, and fluorescent standards for use in such methods.

BACKGROUND OF THE INVENTION Fluorometry is the measurement of fluorescence. It is a valuable analytical tool for quantitative and qualitative analysis. Fluorescence, in turn, involves the molecular absorption of light energy at one wavelength and its instantaneous re-emission at another, usually longer, wavelength. Some molecules fluoresce naturally, while others lend themselves to the formation of a fluorescent compound. Fluorescent compounds have two characteristic spectra: an excitation spectrum (the wavelength and amount of light absorbed) and an emission spectrum (the wavelength and amount of light emitted). These spectra are often referred to as a compound's fluorescence signature or fingerprint. No two compounds have the same fluorescence signature. It is this principle that makes fluorometry a highly specific analytical technique.

The instrument used to measure fluorescence is called a fluorometer. A fluorometer generates the wavelength of light required to excite the analyte of interest, selects the wavelength of light emitted, then measures the intensity of the emitted light. The emitted light is proportional to the concentration of the analyte, up to a maximum concentration. Fluorometers employ either monochromators or optical filters to select excitation and emission wavelengths. Fluorometers that use optical filters for wavelength selection are called filter fluorometers. Fluorometry is chosen for its extraordinary sensitivity, high specificity, simplicity, and low cost. It is a widely accepted, powerful technique that is used for a variety of environmental, industrial, and biotechnology applications. Limits of detectability depend to a large extent on the properties of the sample being measured. Using a fluorometer, detectability is usually about 0.1 part per billion, but can be below several parts per trillion. This is 1,000 to 500,000 times lower than the detection limit of a spectrophotometer. This extraordinary sensitivity allows the use of smaller sample sizes and in vivo measurements. In terms of specificity, spectrophotometric techniques are especially prone to interference problems because many materials absorb light. Fluorometry is highly specific and less susceptible to interferences because fewer materials absorb and emit light (fluoresce). In the event any non-target compounds also absorb and emit light, it is rare that they will emit the same wavelength of light as target compounds.

Such approaches are generally also amenable to samples having a wide concentration range. Fluorescence is linear to (i.e., corresponds directly with) sample concentration over a very large range. Hence, fluorometry can often be used over three to six decades of concentration without sample dilution or modification of the sample cell.

Fluorometry is also a relatively simple analytical technique. Fluorometry's sensitivity and specificity serve to reduce or eliminate the sample preparation procedures often required to concentrate analytes or remove interferences from samples prior to analysis. This reduction in, or elimination of, sample preparation time not only simplifies, but also expedites the analysis. Reagent and instrumentation costs are low when compared to many other analytical techniques. Less reagent can be used due to fluorometry's high sensitivity and small laboratory filter fluorometers can be purchased for less than $5,000 US.

The key components of a filter fluorometer typically include a lamp, excitation filter, sample cuvette, emission filter, and light detector. The lamp or light source provides the energy that "excites" the compound of interest. The lamp emits a broad range of light, more wavelengths than those required to excite the compound.

The excitation filter is used to screen out the wavelengths of light not specific to the compound being measured. This filter allows a smaller band of light energy to pass through and excite the sample. The cuvette or sample cell holds the sample of interest. The cuvette material must allow the compound's absorption and emission light energy to pass through. Also, the size of the sample cell affects the measurement. The greater the pathlength (or diameter) of the cell, the lower the concentration that can be read. Stray light and background fluorescence is also emitted from the sample cell. The emission filter screens out these components, allowing only wavelengths of light specific to the compound to pass through.

The light detector is typically a photomultiplier tube or photodiode. The light passing through the emission filter is detected by the photomultiplier tube. The light intensity, which is linearly proportional to the compound's concentration, can be registered as a digital readout.

Fluorescence applications are varied, and include chlorophyll measurement, algae monitoring, fluorescent tracer studies, the detection and monitoring of aromatic hydrocarbons in water, mud logging, brightening agent measurement, DNA/RNA quantitation, protein quantitation, enzyme activity, cell proliferation, histamine analysis, vitamin assays, aflatoxin analysis, green fluorescent protein (GFP) quantitation, antibiotic sensitivity testing, and the analysis of bacterial viability.

For instance, fluorometry is used to measure chlorophyll, the photosynthetic pigment present in all forms of algae. Chlorophyll determinations are made on lakes, rivers, reservoirs and ocean waters, enabling scientists to draw conclusions on a water body's health, composition and ecological status. Fluorometry is also used for the continuous, on-line detection of algae at drinking water and wastewater facilities.

In tracer studies, fluorometry, in conjunction with a nontoxic, EPA-approved fluorescent dye, is used to measure water flows, to study and model surface and groundwater systems, to trace contaminants in emergency response situations, to detect leaks, and to measure tank retention times.

For detecting and monitoring aromatic hydrocarbons in water, fluorometry can be used to monitor and detect benzene-toluene-ethylbenzene-xylene (BTEX), gasoline, diesel, jet fuel, crude oil, aromatic solvents, and refined petroleum products containing aromatics. Detection limits range from low parts per billion (e.g., on the order of μg/L) to high parts per million (e.g., on the order of mg/L). Applications include emergency response, pollution prevention, leak detection, regulatory compliance, carbon break-thru, treatment verification, process control, discharge monitoring, and upset monitoring.

In the course of "mudlogging", for instance, Texaco has developed and patented a Quantitative Fluorescence Technique (Q.F.T. ™) which can be used to accurately measure the relative amount of petroleum in drill colonies. In other applications, fluorometry can be used to detect and measure brightening agents commonly found in laundry wastewater. High levels of brightening agents can indicate improper wastewater discharge. The quantitation of DNA and/or RNA can be a prelude to many practices in molecular biology. Fluorometric analysis with one of many alternative fluorescent dyes is used for the reliable and accurate quantitation of DNA. The high sensitivity and selectivity of fluorometric assays over spectrophotometric methods has led to the development of instruments designed to quantitate nucleic acids based on their interactions with fluorescent dyes.

Fluorometry is also used for protein quantitation, and the analysis of enzyme activity and cell proliferation. Fluorometry can be used with commercial assays for accurate quantitation of proteins in solution, for enzyme activity studies, or for sensitive cell counting and proliferation. Fluorometry's high selectivity makes it a popular technique for histamine analysis both in biological research and as a quality control measure in the fish industry. Quantitative determinations of vitamins is important in the food and pharmaceutical industries. Fluorometric analysis is often chosen over other quantitation methods due to its sensitivity, specificity, and simplicity. Fluorometry is also a proven, simple and accurate technique for the determination of low levels of aflatoxin in corn, rice, peanuts, peanut butter, tree nuts, cottonseed, feed, and grain samples. Similarly, the green-fluorescent protein from the jellyfish Aequorea victoria is used as a fluorescent marker for gene expression in a variety of organisms ranging from bacteria to higher plants and animals. Antibiotic sensitivity methods using fluorescent probes reduce the test-to-result period from up to 48 hours and allow bacterial viability to be continually monitored in real time. In terms of bacterial viability, a novel two-color fluorescence assay that allows researchers to quantitatively distinguish live and dead bacteria in minutes, even in a mixed population containing a range of bacterial types. The assignee of the present invention, Turner Designs, Inc. offers a variety of different filter fluorometers, each designed to meet a particular need. These include:

The "10-AU" Digital Field Fluorometer, which is a rugged, field-portable instrument that can be set up for continuous-flow monitoring or discrete sample analysis. A watertight case, internal data logging, automatic range changing, and stability make the 10-AU particularly suited for field studies.

The "TD-700" and "TD-3100" Laboratory Fluorometers, which are both compact, benchtop fluorometers having a multi-assay filter cylinder, menu-driven software, an RS-232 serial interface, and multi-point calibration.

The "TD-360" Mini-fiuorometer, which is a variable-wavelength filter fluorometer designed for quick, easy, and accurate fluorescence measurements. The multi-wavelength capability makes it appropriate for a wide variety of molecular biology applications. A removable optical filter holder and user-changeable light source allow the quantitation of assays of differing wavelengths using a single instrument. The "TD-4100" continuous on-line "oil-in-water" (e.g., aromatic hydrocarbons in water) monitor, designed for use as a low-maintenance, non-fouling, on-line monitor for hydrocarbons in water. BTEX, gasoline, diesel, jet fuel, crude oil, aromatic solvents, and refined petroleum products containing aromatics can be detected and monitored. The "TD-4100 XD" is a more rugged version of the TD-4100 continuous online oil in water monitor, designed specifically for offshore oil production, refining, petro-chemical, mining, and other industries that require robust on-line hardware for severe duty and hazardous area locations.

The use of such instruments typically requires that they be calibrated by the use of standards. "Primary" standards can occasionally be used, in the form of one or more known concentrations of the particular analyte of interest. Preparing primary standards for fluorometry can be challenging, as well as time consuming. Oftentimes, storage of primary standards is difficult because of special storage conditions (e.g., temperature) that may affect the standards' viability over time. In addition, the preparation and/or use of primary standards can lead to difficulties with the assay, e.g., in view the possibility of chemistry errors, instability of stock standards, and instability of primary standards. Much time can be wasted in trying to determine where the error occurred. For these, and other reasons, the use of "secondary" standards has evolved. Oftentimes, in the analysis of petroleum hydrocarbons in water, primary standards do not exist. In the case of industrial wastewater, the complex mixture of hydrocarbons from a refinery affluent precludes the determination or creation of a primary standard. Fluorometers are calibrated by correlating their response to standard laboratory methods. The solid standard can then be used to "spike" or quality control check the performance of the instrument (i.e., check electronic drift). Typically, a secondary standard involves the use of a material other than the analyte of interest, which approximates the fluorescence of the analyte. The standard is ideally one that can be packaged in a known concentration and sold, stored and used in a stable form. Examples of such standards include "solid" standards, in the form of solid or semi-solid polymeric materials (e.g., polymethyl methacrylate rods) that have been doped with the fluorescent material of interest. Solid standards are commercially available and are commonly used to both calibrate the wavelength of fluorometers and to determine their sensitivity.

For instance, the fluorometric analysis of chlorophyll a is commonly done with environmental and other samples. Unfortunately, chorophyll is quite unstable in solution, making repeated calibrations with chlorophyll itself both expensive and time consuming. Fortunately, however, once a given fluorometer has been calibrated, a secondary standard can be used, having reasonably similar spectral characteristics to the analyte of choice. The secondary standard is read at the time of initial calibration against the analyte (e.g., chlorophyll), and its reading (which may even be on a different scale) is noted. Thereafter, the fluorometer can be checked and adjusted solely by use of this stable secondary standard. Turner Designs, for instance, recommends the use of coproporyphin as a secondary standard, which is relatively stable and fluoresces in the same wavelength as chlorophyll. Conventionally, such standards were prepared in acid solutions and used with considerable caution.

A scaled series of eight white scale fluorescence standards is available from Labsphere, Inc. North Sutton, NH. The standards sets are available in glossy or matte finished with a 2.00 D reflective area. Similarly Hitachi (Hitachi Instruments, Inc., Nissei Sangyou Co. Ltd., San Jose, CA) has available a variety of standard samples that can be used to perform wavelength calibration and to monitor the performance of fluorescence spectrophotometers. A kit includes a series of polymethylmethacryate (PMMA) standards containing fluorophores in various concentrations, each standard corresponding to a unique excitation and emission maximums.

Photon Technology International sells solid fluorescent standards for calibration, spectral resolution evaluation, and the like. The standard is described as a single crystal of dysprosium-activated yttrium aluminum garnet. When excited by UF (from 250 to 500 nm) the crystal emits high intensity light simultaneously in four bands in a range from 450 to 800 nm.

Commercially available secondary standards tend to suffer from a number of drawbacks, however, including the fact that they tend to lack precision. Typically, the emission levels of such standards are determined, and varied to the extent possible, by altering the amount of fluorophore incorporated therein. While this works for conventional purposes, such as adding a fluorescent dye to water as a quality control check standard, this approach fails to provide such features as precision or controllability. This is particularly the case where human error can arise in the course of preparing the standards themselves, and can be exacerbated in applications where the operator is expected to perform only routine, predictable steps, and is not particular trained in chemistry or analytical problem-solving. BRIEF DESCRIPTION OF THE DRAWING In the Drawing:

Figure 1 shows an exploded perspective view of a preferred fixed standard of the present invention.

Figure 2a and 2b show exploded perspective views of a preferred adjustable standard of the present invention.

SUMMARY OF THE INVENTION In a preferred embodiment, the present invention provides an apparatus for use as a spectrophotometric (e.g., fluorometric, UV or IR) standard, the apparatus comprising a housing adapted to be placed within a spectrophotometer (e.g., fluorometer) and containing a stable standard material (e.g., in the form of a dye- doped polymer, solid crystal, or mirror to create scattered light). The housing, in turn, comprises one or more apertures configured to provide a predetermined absorption, excitation and or emission level in the course of using the standard within the particular device. The aperture(s) can, independently, be either fixed or adjustable, and of the same or different dimensions and orientation with respect to the fiber. One or more fixed apertures can be used to provide a precise and predetermined emission level, while adjustable apertures can be used to permit the operator to select and control the emission levels at the time of use, e.g., in the field. In turn, standards of the present invention can be used in ways not generally possible with conventional standards, e.g., by providing them with precise, predetermined, and optionally controllable, emission levels. Standards of the present invention provide an optimal combination of such properties as uniformity, stability, adaptability, cost and ease of use.

In another aspect, the invention provides a combination comprising a fluorometric standard of the present invention positioned within a fluorometer. In yet another aspect, the invention provides a kit comprising a plurality of fluorometric standards, each providing a predetermined excitation and/or emission level. The standard apparatus can be used as standards in any single tube (one sample analysis at a time) fluorometer, preferably by providing universal housings and/or by adapting the housing for the particular instrument. The concentration of light emitted from the rod is dependent on the amount of light exciting (i.e., impacting) the rod. To control the amount of excitation energy reading the rod, the manufacturer and or user can alter slit sizes of the holder, or move an adjusting mechanism (e.g., ajustable screw or rod adapted to be used with the polymeric rod) to cover and/or expose respective portions thereof.

In other aspects, the invention provides methods of preparing and using solid fluorometric standard apparatuses as described herein.

DETAILED DESCRIPTION When conducting fluorometric studies, secondary standards of the present invention provide a simple, stable, and less expensive alternative to multiple calibrations with a primary standard. Once the instrument is calibrated with a primary standard, the secondary standard can effectively be used to check the stability of the instrument on a periodic (e.g., daily) basis. The standard of the present invention can also be used for calibration in place of the primary standard, with only occasional verification of results using a primary standard or secondary method, for example, by correlating fluroescence to the results of an accepted (e.g., EPA) method such as a gravimetric or gas chromatographic method. Thus, monitoring instrument drift and routine recalibrations can be performed with standards of the invention as well.

The present invention will be described with respect to a preferred embodiment in which a fluorescent standard is used to calibrate a fluorometer, although it is understood that the invention has application to other uses as well, including for use as ultraviolet and infrared absorption standards in connection with corresponding spectrometers.

A fixed emission standard apparatus of the present invention can be provided in any suitable form, e.g., as a solid or semi-solid material positioned with an impervious (e.g., opaque) housing, the housing, in turn, having one or more apertures (e.g., in the form of slits, holes, or a mesh- like design) designed to permit a constant and predetermined amount of fluorescence to be detected, whether by controlling the excitation or detection of the sample, or both.

An adjustable emission standard apparatus can also be provided in any suitable form, e.g., in the form of one or more adjustable portions located between the standard source and other components of the fluorometer. The adjustable portions can be selectively adjusted to limit or control either the amount of excitation light entering the apparatus and/or the amount of emission fluorescence exiting it, thereby providing a means of "fine tuning" the emission level of the apparatus. Such control can be achieved in any suitable way, e.g., with manual or electronic controls, or automatically. Optionally, an adjustable aperture apparatus can also be included as a discrete component positioned within the emission path between the standard and the detector, without changing the emission level or distribution of the standard source itself. The adjustment of incoming excitation light, or exciting emissions can be accomplished in any suitable fashion, e.g., by the use of diaphragm-like apertures, mutually rotatable slits and solid components, a slit-like blind mechanism, or a rolling blind.

Adjustment can also be achieved by moving (e.g., rotating) the solid standard holder relative to the detector and/or excitation source, e.g., in order to focus more or less light on the detector. In an alternative approach adjustment can be achieved by positioning or re-positioning one or more slits, e.g., to position it off the axis of the detector of excitation source.. In yet another embodiment, adjustment can take the form of a mechanism for altering the distance between the aperture (e.g., slit) and/or the solid standard, the detector, and/or the excitation source, though recalling that increasing such distances tends to decrease the signal received.

In a related embodiment, emission can be controlled and adjusted, for instance, by the use of plurality of slits positioned between the source and the detector in such a manner that the slits can be controlled in a manner that varies either the effective number of slits, their absolute and relative degrees of angular orientation (tilt), and/or the spacing between them.

A standard of the present invention, in the form of a fluorometric standard, will typically be adapted for use in combination with a fluorometer having the following components. In one embodiment, the present invention provides a standard positioned within such a fluorometer.

The fluorometer will include a lamp or light source to provide the energy necessary to "excite" the compound of interest. The lamp emits a broad range of light, more wavelengths than those required to excite the compound. An excitation filter is used to screen out the wavelengths of light not specific to the compound being measured. This filter allows a smaller band of light energy to pass through and excite the sample. A cuvette or sample cell holds the sample of interest, and in this case, the standard housing itself. The sample cuvette material must allow the compound's absorption and emission light energy to pass through. Also, the size of the sample cell affects the measurement. The greater the pathlength (or diameter) of the cell, the lower the concentration that can be read. By the use of an emission filter, stray light and background fluorescence is also emitted from the sample cell. The emission filter screens out these components, allowing only wavelengths of light specific to the compound to pass through. A light detector will typically take the form of a photomultiplier tube or photodiode. The light passing through the emission filter is detected by the photomultiplier tube. The light intensity, which is linearly proportional to the compound's concentration, is registered as a digital readout.

An apparatus of the present invention will be described with reference to the Drawing. As may be seen from the following description and accompanying Drawing, the present invention contemplates several basic embodiments and configurations of the apparatus, which can further include various modifications and options as well. In the description of the several figures, like elements in the Figures are identified by like numerals.

Figure 1 shows a preferred embodiment of a fixed emission secondary standard apparatus (10) of this invention, including a housing component (12) and fluorescent fiber standard component (14). The housing is provided in a generally rectangular configuration, having major walls surfaces (16a and 16b being shown), and solid top and bottom portions 17a and 17b, respectively. The housing is dimensioned to fit within a desired instrument, e.g., by generally assuming the shape of cuvettes used in such an instrument. In the embodiment shown, each of the four major wall surfaces includes a corresponding emission slit, (18a and 18b being shown), aligned centrally along the face of the corresponding wall, and longitudinally parallel with a central bore 20 formed within the housing, the bore being adapted to receive fluorescent fiber 14. The housing is also provided with integral tab 22, or other suitable manipulating features, which can be used to grasp and move the housing portion and which further facilitates the precise and accurate measurement of the solid standard measurement.

The bore 20, in turn, is provided having dimensions (particularly diameter and depth) adapted for the particular fiber standard of choice. The bore and apertures can be formed in any suitable manner and location, in the course of fabricating (e.g., molding) the housing itself, or thereafter, by the use of conventional tooling with a previously fabricated piece. The fiber is itself dimensioned and adapted (e.g., in terms of diameter, length and stiffness) to be positioned fully and uniformly within the bore, and there secured by suitable means, such as epoxy 24, followed by placement and secured attachment of the housing base 17b.

As shown, the apparatus is assembled by positioning a standard-containing rod within the central bore, though it is understood that alternative embodiments can take a variety of forms, for instance, forms in which the bore is not in a central location with respect to the housing. The rod can be permanently secured within the bore, e.g., by the use of adhesive, or can be removably positioned therein, e.g., snugly fit in order to permit reuse of either component.

Figures 2a and 2b show an exploded perspective views of a preferred embodiment of an adjustable standard apparatus 30 of this invention. In Figure 2a, the apparatus 30 is shown having a generally cylindrical housing portion 32 adapted (e.g., dimensioned) to be used with a particular spectrometer of interest. The housing includes one or more orientation means 32 (e.g., pegs), to facilitate the proper positioning and seating of the housing within the corresponding instrument. The housing further includes an integral graspable upper portion 36 adapted to be grasped by the user in order to insert, remove, and/or position the housing. The housing is provided in the form of a housing assembly that further includes a hexnut wrench 38, for use in securing the standard within the housing. The housing, in turn, is secured within the instrument by any suitable means, e.g., by using one or more recessed clips 39 within the outer wall of the housing to provide a compressed fit between the housing and the opposing wall of the instrument chamber. The apertures can also be of the same or different dimensions as they traverse the body of the housing, for instance, they can taper down in size from the outer surface toward the housing bore, in order to their fabrication and/or in order to improve optical efficiency. Under such circumstances, the smallest aperture dimension would typically control the amount of excitation/emission passing through the aperture. The housing assembly of Figure 2a further includes a holder portion 40 adapted to be secured to an outer wall of the instrument by means of associated double sided tape strips 42. The holder portion includes a cavity adapted to mate with and securely hold the housing portion (typically itself having a standard inserted therein) when not in use. Turning briefly to Figure 2b, there is shown an exploded perspective view of the housing portion and standard component 46, which is shown in exploded view as well as including the fluorescent fiber 48 and hexnut 50, secured together by means of a suitable adhesive interface (not shown). The hexnut/fiber combination can be used to adjust the apparatus by rotating the hexnut, thereby extending or retracting the fiber within the bore, thereby varying its position with respect to the adjacent aperture(s). The excitation/emission apertures can be of the same or different dimensions as well, thereby providing further options with regard to its use. These and other features can be modified in any suitable manner, in order to adapt the fixed of adjustable standards to the specific requirements (e.g., cuvette holder dimensions and placement) of any fluorometer. A particularly preferred embodiment, for instance, is adapted for use in the fluorometer identifed as Model 4100 available from Turner Designs, Sunnyvale, CA.

The housing component of a standard of the present invention can be fabricated from any suitable material or combination of materials. Suitable materials provide an optimal combination of such features as machinability, durability, lack of interference with the fluorescence or other signal generated, and resistance to decay by UN, IR, and visible light. Materials useful for such purposes will become apparent to those skilled in the art, given the present description, and include polymeric materials such as polystyrene (available under the tradename "Delrin"), and metals such as aluminum.

The standard itself similarly provides an optimal combination of such features as stability, ease of use, and cost. Examples of suitable standards include, for instance, fluorescent fibers as are available from Bicron Corporation, Νewbury, Ohio. Commercially available fibers include those identified as "BCF-93" (green), "BCF- 99-06A " (orange), and "BCF-99-172" (red).

Those skilled in the appropriate art will also be able to identify one or more fluorescent materials (e.g., dyes) for use in analyzing a wide variety of analytes. Examples of suitable analytes and dyes expected to be used in connection with the standards and methods of the present invention include, but are not limited to: Aflatoxin, alkaline phosphatase (4-methylumbelliferyl phosphate), B- galactosidase, chlorophyll a, DiFMUP (6,8-difluoro-4-methylumbelliferyl phosphate), proteases (e.g., EnzChek(TM) Protease), ethidium bromide, FITC, fmorescein, GFPwt (Green Fluorescent Protein), histamine (phthalaldehyde), Hoechst Dye 33258 (DΝA), MUP (4-methylumbelliferyl phosphate), ΝADPH, ΝanoOrange(TM), PicoGreen(R), Rhodamme WT, RiboGreen(TM), SYTOX(R) Green, and Thiazole Orange.

Similarly, the dimensions, orientation and number of slits can be varied according to the desires of the manufacture and/or user. Standards of the present invention can be adapted for use with most, if not all, commercially available spectrometers (e.g., fluorometers). For instance, non-adjustable standards having 2 mm fluorescent fibers can be used in fluorometers having model numbers 10-AU, model 10, TD-700 and TD-360 fluorometers, while 3 mm or 5 mm adjustable standards can be used in the TD-3100 and the TD-4100 (and TD-4100XD) model fluorometers, each available from Turner Designs, Sunnyvale California. The larger rod, in turn, permits a wider slit to be used in the holder, which in combination with the adjustable feature, permits a larger fluorescent signal to be generated as compared to the 2 mm rod.

In a preferred embodiment, the present invention provides a kit comprising a plurality of standards (e.g., 4, 6 or 8 standards) which together can be used to cover the full wavelength range (or any desired portion thereof) of a particular fluorometer and/or application.

In one embodiment, a general method of using a fluorometric standard of the present invention involves the steps of:

1. Calibrating the fluorometer with known liquid standard. 2. Inserting the secondary standard to obtain value.

3. Running samples (unknowns) in the recommended manner.

4. Secondary standard can be inserted at anytime to check the stability of the instrument. The operator should expect a value close to that obtained in step 2. If not, the instrument may need to be recalibrated with the secondary standard to achieve the value obtained in step 2.

The option of adjustability provides new and unique advantages in the use of standards, as described in the following general method:

1. A laboratory fluorometer (e.g., TD-3100, Turner Designs) is used that will support calibrated and uncalibrated methods for measuring fluorescent hydrocarbons in water or soil. These methods may involve directly measuring hydrocarbons in water without solvent extraction, or involve measuring hydrocarbons in solvents (i.e., hexane) used to extract targeted hydrocarbons from water or soil. Regardless of which method is used to measure targeted hydrocarbons; incorporating a stable reference standard (or QC standard) as part of the method will improve the precision and accuracy of the hydrocarbon analysis. A non-liquid, solid standard of the present invention is provided as a stable reference standard, QC standard or secondary standard. The standard is provided in the form of a 25mm holder that contains a fiber which will produce a fluorescent signal within an excitation and emission operating range of 254-410 nm excitation and 300-500 nm emission. The intensity of the fluorescent signal produced by the standard can be set by the user and is attenuated (tuned) buy adjusting a set screw. The standard can be attenuated to operate at any sensitivity setting used for the instrument. Additional preferred performance features of the standard include:

1. Shelf-life greater than two years. 2. Operational performance that is unaffected by changes in laboratory temperature.

3. Simple set-up for sustained performance across the complete sensitivity range of the fluorometer

A standard kit is provided and opened at the time of use, the kit including the standard itself (including housing and fluorescent fiber), hexnut screwdriver, calibration record, and standard holder. The hexnut screwdriver is used to adjust or tune the standard to calibrate the fluorometer. The calibration record is used to record the fluorescent signal of the standard as required by an analytical method. The standard holder is designed to attach to the side of the fluorometer and is used to store the standard when not in use. Tuning the Solid Standard 1. Set-up and establish the basic sensitivity of the fluorometer as defined by the analytical method designed by the operator or manufacturer to measure targeted hydrocarbons in water or in solvent extracts. 2. Remove all adapters from the filter cylinder.

3. Insert the standard into the filter cylinder so that the dot on top of the standard is property oriented. Close the lid to the fluorometer and allow the reading to stabilize for 10 seconds, then press the appropriate key to read the standard fluorescence signal. 4. The ideal setting for the standard fluorescence output is approximately 50% of the full scale display for raw fluorescence readings or the mid-point for calibrated readings from the instrument, when using a "TD-3100" fluorometer. Whether the standard is being used as a reference standard, quality control standard or secondary standard; tuning the standard to operate in the mid range of the analytical method will provide the highest degree of analytical precision for any method.

5. If the standard reading is too high, then remove the standard and rotate the hexnut clockwise to decrease the fluorescent output of the standard when re- inserted into the filter cylinder. If the value is too low, then remove the standard and rotate the hexnut counter clockwise to increase the fluorescent output of the standard when re-inserted into the filter cylinder. Repeat steps 3- 5 until the optimal value is obtained for the standard to match the needs of the particular analytical method for a reference standard, quality control standard or secondary standard.

6. Record the final value for the standard on the calibration record.

7. Proceed with analyzing your samples.

8. The standard should be measured frequently to insure that the instrument performance is stable. If the fluorescence output from the standard no longer matches its recorded value, and exceeds the recorded value by a percentage greater than the analytical precision of the method, then the instrument should be recalibrated or sensitivity reset to match the signal output of the standard as defined in the calibration record.

2199204

Claims

CLAIMSWhat is claimed is:

1. A spectrophotometric standard apparatus comprising a housing adapted

to be placed within a selected spectrophotometer, the housing containing a stable

standard material and comprising one or more apertures configured to provide

predetermined absorption, excitation and/or emission levels within the

spectrophotometer.

2. An apparatus according to claim 1 wherein the spectrophotometer is a

fluorometer and the standard material is a fluorometric standard.

3. An apparatus according to claim 2 wherein the standard material

comprises a dye-doped polymer.

4. An apparatus according to claim 3 wherein the standard material is

provided in the form of a fluorescent fiber.

5. An apparatus according to claim 2 wherein the housing comprises one

or more fixed apertures.

6. An apparatus according to claim 2 wherein the housing comprises one

or more adjustable apertures.

7. An apparatus according to claim 5 wherein the fixed aperture(s)

provide a precise and predetermined emission level.

8. An apparatus according to claim 6 wherein the adjustable aperture(s)

are adapted to permit an operator to select and control excitation and/or emission

levels at the time of use.

9. An apparatus according to claim 7 wherein the standard material is

provided in the form of a solid or semi-solid material positioned with an opaque

housing having fixed apertures in the form of slits to permit a constant and

predetermined amount of fluorescence to be detected.

10. An apparatus according to claim 8 wherein the standard apparatus

comprises one or more adjustable portions located between the standard source and

corresponding excitation/detection components of the fluorometer, the adjustable

portions adapted to be selectively adjusted to limit or control either the amount of

excitation light entering the apparatus and/or the amount of emission fluorescence

exiting the apparatus.

11. An apparatus according to claim 10 wherein the adjustable portions are

adjusted by means selected from the group consisting of manual adjustments and

electronic controls.

12. An apparatus according to claim 4 wherein the fluorescent fiber is

selected from the group consisting of 2 mm, 3 mm and 5 mm diameter fluorescent

fibers.

13. A combination comprising a standard apparatus according to claim 1

positioned within a spectrophotometer.

14. A combination according to claim 13 wherein the spectrophotometer is

a fluorometer and the standard material is a fluorometric standard.

15. A combination according to claim 14 wherein the standard material

comprises a dye-doped polymer and is provided in the form of a fluorescent fiber.

16. A combination according to claim 15 wherein the housing comprises

one or more fixed apertures.

17. A combination according to claim 15 wherein the housing comprises

one or more adjustable apertures.

18. A kit comprising a plurality of standard apparatuses, each according to

claim 1, and each providing a predetermined excitation and/or emission level.

19. A kit according to claim 18 wherein the standard materials are each

fluorescent materials.

20. A kit according to claim 19 wherein the plurality of standard materials

are adapted to cover a predetermined wavelength range.

21. A method of preparing a fluorometric standard apparatus according to

claim 2, wherein the method comprises positioning a fluorescent fiber within an

opaque housing having one or more apertures adapted for effecting and/or detecting

the excitation and/or emission of the fibers when positioned within a fluorometer.

22. A method according to claim 21 wherein the apertures comprise fixed

apertures in the form of slits to permit a constant and predetermined amount of

fluorescence to be detected.

23. A method according to claim 21 wherein the apertures comprise

adjustable apertures adapted to permit an operator to select and control excitation

and/or emission levels at the time of use.

24. A method of using a fluorometric standard apparatus, wherein the

method comprises providing an apparatus according to claim 2, positioning the apparatus within a fluorometer, and using the standard apparatus to calibrate and/or

determine the stability of the fluorometer.

25. A method according to claim 22 wherein the apparatus comprises one

or more fixed apertures in the form of slits to permit a constant and predetermined

amount of fluorescence to be detected.

26. A method according to claim 22 wherein the apparatus comprises one

or more adjustable apertures adapted to permit an operator to select and control

excitation and/or emission levels at the time of use.