CA1113279A - Colorimetric analytical test apparatus - Google Patents
Colorimetric analytical test apparatusInfo
- Publication number
- CA1113279A CA1113279A CA330,984A CA330984A CA1113279A CA 1113279 A CA1113279 A CA 1113279A CA 330984 A CA330984 A CA 330984A CA 1113279 A CA1113279 A CA 1113279A
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- Prior art keywords
- zone
- test apparatus
- adsorbent
- test
- glucose
- Prior art date
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
- G01N33/521—Single-layer analytical elements
- G01N33/523—Single-layer analytical elements the element being adapted for a specific analyte
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/54—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving glucose or galactose
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- Life Sciences & Earth Sciences (AREA)
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- Organic Chemistry (AREA)
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- Proteomics, Peptides & Aminoacids (AREA)
- Molecular Biology (AREA)
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- General Health & Medical Sciences (AREA)
- Hematology (AREA)
- Biotechnology (AREA)
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- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
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- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Urology & Nephrology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Emergency Medicine (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
TITLE
COLORIMETRIC ANALYTICAL TEST APPARATUS
INVENTOR
Douglas Ian McGregor ABSTRACT OF THE DISCLOSURE
An analytical test apparatus suitable for the colorimetric determination of glucose in the presence of inhibitors includes:
- an inert solid backing having a sample-receiving zone (a) spaced from an adsorbent zone (b) and test zone (c), - a wick to transport liquid from zone (a) to zone (b), - activated carbon adsorbent at zone (b), and - glucose test paper at test zone (c);
the three zones being in liquid-conducting capillary contact.
The amount and nature of the adsorbent is selected to preferentially remove the inhibitors but not the glucose.
The apparatus is especially useful as a stick for determining glucosinolate contents of oilseeds such as rapeseed. A kit including this test apparatus is also described.
COLORIMETRIC ANALYTICAL TEST APPARATUS
INVENTOR
Douglas Ian McGregor ABSTRACT OF THE DISCLOSURE
An analytical test apparatus suitable for the colorimetric determination of glucose in the presence of inhibitors includes:
- an inert solid backing having a sample-receiving zone (a) spaced from an adsorbent zone (b) and test zone (c), - a wick to transport liquid from zone (a) to zone (b), - activated carbon adsorbent at zone (b), and - glucose test paper at test zone (c);
the three zones being in liquid-conducting capillary contact.
The amount and nature of the adsorbent is selected to preferentially remove the inhibitors but not the glucose.
The apparatus is especially useful as a stick for determining glucosinolate contents of oilseeds such as rapeseed. A kit including this test apparatus is also described.
Description
2~
This invention is directed to an analytical test apparatus for a simple, rapid and reliable colorimetric determination of ~lucose in compositions which contain inhibitors of the desired colour development due to glucose.
The invention is particularly useful for the determination of glucosinolates in oilseeds and oilseed meals, the glucosinolates being enzymatically hydrolyzed to glucose before assay. A kit including the test apparatus for determinations in the field, is described.
Background and Prior Art The determination of glucose is widely practiced since this is the sugar which diabetics must monitor in their body fluids. Glucose-specific test papers have been developed ; and are on the market. One commonly used test paper contains I two enzymes (glucose oxidase and peroxidase~, the chromogen o-tolidine, and a yellow dye. When an aqueous solution of glucose comes in contact with this paper, hydrogen peroxide ` is produced by the action of glucose oxidase on glucose. The resulting hydrogen peroxide in the presence of o-tolidine and peroxidase forms a blue complex. Against the background - of the yellow dye, the test paper appears green (the higher the glucose concentration the more intense the green colour) - and a colorimetric determination can be made. Inhibitors of these colour-forming reactions are not normally encountered in body fluids but may be encountered in other systems.
Glucosinolates are a family of sulfur-containin~
compounds, at least nine of which occur in the seed of rapeseed (Brassica napus L. and Brassica campestris L.) and other oilseeds such as mustard and crambe. They are ` 30 undesirable compounds because their hydrolysis products, isothiocyanates, thiocyanates, nitriles and epithionitxiles ~ '.
- - .
2~
(aglycones) cause metabolic upsets in non-ruminant animals.
Their presence in rapeseed is a major constraint on more :intensive and widespread use of rapeseed meal in animal feeds and potentially could limit the use of protein isolates or concentrates for human consumption. Plant breeders, therefore, have concentrated efforts on developing varieties reduced or free of glucosinolates. The recent introduction in Canada of new varieties of rapeseed such a~ Tower, Regent, Altex and Candle with substantially reduced levels of glucosinolates, has created a preference for low glucosinolate-containing seed and meal in both the domestic and international markets. ~-However, these markets can only be served if the new low glucosinolate-containing seed and meal is kept separate from high glucosinolate-containing seed and meal throughout the production, processing, transportation and marketing system.
This is difficult because high and low glucosinolate-containing seed and meal are visually indistinguishable.
Production and marketing would be greatly facilitated if there were a better method available which was suitable for use at all points in the production and marketing system to distinguish low glucosinolate rapeseed or meal.
Analytical methods have been devised for determining levels of glucosinolates by measuring inorganic sulphate (released on hydrolysis) or the other aglycones. ~ -However, since the procedures for these quantitative analyses are time-consuming, technically demanding, and require expensive chromatographic and spectrophotometric equipment, they are of limited usefulness in commercial trade for segregating seed according to glucosinolate content, and their application has been confined to selection in plant breeding and to control of pedigree seed lots.
.
;.. . .
. . ~ . , -::
2~9 The first to demonstrate that glucose test papers could be used to determine the approximate glucosino-late level in rapeseed was K.A. Lein (Z. Pflanzenzuecht 63, 137-154, 1970). In 1972, Bjorkman reported the use of glucose oxidase, peroxidase and the chromogen o-dianisidine to colorimetrically estimate in solution the quantity of purified glucosinolates from rapeseed extracts. However, when such colorimetric analyses were applied to crude extracts of glucosinolate-containing Cruci~erae including rapeseed, interfering substances were found to inhibit colour develop-ment (Van Etten et al, J. Agric. Food Chem. 22, 483-487, 1974).
These workers found that the inhibition could be overcome by treating the extracts with charcoal before colorimetric analysis or, when using glucose test paper, by separating the inhibiting substances from glucose in an aqueous seed extract through capillary action up a strip of test paper.
For rapid screening with test paper, Van Etten et al considered the capillary separation up the test paper the most satis-factory.
; 20 ~ However, for enhanced sensitivity and reproducibility when rapeseed extrac's were being assayed, ; McGregor and Downey (McGregor, D.I. and Downey, R.K., A Rapid and Simple Assay for Identifying Low Glucosinolate Rapeseed, Can. J. Plant Sci. 55, 191-196, 1975) found that the addition of charcoal was necessary. In the absence of charcoal, color development of the glucose test paper with crude rapeseed ^' extracts was variable and mottled such that quantitative ~ . . , analysis at best was dif~icult if not impossible. The controlled addition of activated carbon powder, resulted in uniform color development over the whole test paper strip
This invention is directed to an analytical test apparatus for a simple, rapid and reliable colorimetric determination of ~lucose in compositions which contain inhibitors of the desired colour development due to glucose.
The invention is particularly useful for the determination of glucosinolates in oilseeds and oilseed meals, the glucosinolates being enzymatically hydrolyzed to glucose before assay. A kit including the test apparatus for determinations in the field, is described.
Background and Prior Art The determination of glucose is widely practiced since this is the sugar which diabetics must monitor in their body fluids. Glucose-specific test papers have been developed ; and are on the market. One commonly used test paper contains I two enzymes (glucose oxidase and peroxidase~, the chromogen o-tolidine, and a yellow dye. When an aqueous solution of glucose comes in contact with this paper, hydrogen peroxide ` is produced by the action of glucose oxidase on glucose. The resulting hydrogen peroxide in the presence of o-tolidine and peroxidase forms a blue complex. Against the background - of the yellow dye, the test paper appears green (the higher the glucose concentration the more intense the green colour) - and a colorimetric determination can be made. Inhibitors of these colour-forming reactions are not normally encountered in body fluids but may be encountered in other systems.
Glucosinolates are a family of sulfur-containin~
compounds, at least nine of which occur in the seed of rapeseed (Brassica napus L. and Brassica campestris L.) and other oilseeds such as mustard and crambe. They are ` 30 undesirable compounds because their hydrolysis products, isothiocyanates, thiocyanates, nitriles and epithionitxiles ~ '.
- - .
2~
(aglycones) cause metabolic upsets in non-ruminant animals.
Their presence in rapeseed is a major constraint on more :intensive and widespread use of rapeseed meal in animal feeds and potentially could limit the use of protein isolates or concentrates for human consumption. Plant breeders, therefore, have concentrated efforts on developing varieties reduced or free of glucosinolates. The recent introduction in Canada of new varieties of rapeseed such a~ Tower, Regent, Altex and Candle with substantially reduced levels of glucosinolates, has created a preference for low glucosinolate-containing seed and meal in both the domestic and international markets. ~-However, these markets can only be served if the new low glucosinolate-containing seed and meal is kept separate from high glucosinolate-containing seed and meal throughout the production, processing, transportation and marketing system.
This is difficult because high and low glucosinolate-containing seed and meal are visually indistinguishable.
Production and marketing would be greatly facilitated if there were a better method available which was suitable for use at all points in the production and marketing system to distinguish low glucosinolate rapeseed or meal.
Analytical methods have been devised for determining levels of glucosinolates by measuring inorganic sulphate (released on hydrolysis) or the other aglycones. ~ -However, since the procedures for these quantitative analyses are time-consuming, technically demanding, and require expensive chromatographic and spectrophotometric equipment, they are of limited usefulness in commercial trade for segregating seed according to glucosinolate content, and their application has been confined to selection in plant breeding and to control of pedigree seed lots.
.
;.. . .
. . ~ . , -::
2~9 The first to demonstrate that glucose test papers could be used to determine the approximate glucosino-late level in rapeseed was K.A. Lein (Z. Pflanzenzuecht 63, 137-154, 1970). In 1972, Bjorkman reported the use of glucose oxidase, peroxidase and the chromogen o-dianisidine to colorimetrically estimate in solution the quantity of purified glucosinolates from rapeseed extracts. However, when such colorimetric analyses were applied to crude extracts of glucosinolate-containing Cruci~erae including rapeseed, interfering substances were found to inhibit colour develop-ment (Van Etten et al, J. Agric. Food Chem. 22, 483-487, 1974).
These workers found that the inhibition could be overcome by treating the extracts with charcoal before colorimetric analysis or, when using glucose test paper, by separating the inhibiting substances from glucose in an aqueous seed extract through capillary action up a strip of test paper.
For rapid screening with test paper, Van Etten et al considered the capillary separation up the test paper the most satis-factory.
; 20 ~ However, for enhanced sensitivity and reproducibility when rapeseed extrac's were being assayed, ; McGregor and Downey (McGregor, D.I. and Downey, R.K., A Rapid and Simple Assay for Identifying Low Glucosinolate Rapeseed, Can. J. Plant Sci. 55, 191-196, 1975) found that the addition of charcoal was necessary. In the absence of charcoal, color development of the glucose test paper with crude rapeseed ^' extracts was variable and mottled such that quantitative ~ . . , analysis at best was dif~icult if not impossible. The controlled addition of activated carbon powder, resulted in uniform color development over the whole test paper strip
-3-. - : . . . , ~
3~3 facilitating quantitative analysis. Color development was enhanced, improving sensitivity so that as little as 10%
by weight contamination of seed with high glucosinolate content/ in seed of low glucosinolate content,could be detected. This test procedure of McGregor and Downey as presently used to identify low glucosinolate rapeseed is simple but somewhat "messy" and slower than desired. It would be difficult to carry out in many field situations. To date use of this assay (McGregor and Downey) has been limited to analytical labs and crushing plants where facilities for handling and disposing of the activated carbon and for cleaning mortar and pestle, etc. are available.
~ or other analyses, various test devices, laminates, sticks, etc. have been developed to simplify and speed up the procedure. Typical prior art devices are described in United States Patents: 3,011,874; 3,993,451;
3~3 facilitating quantitative analysis. Color development was enhanced, improving sensitivity so that as little as 10%
by weight contamination of seed with high glucosinolate content/ in seed of low glucosinolate content,could be detected. This test procedure of McGregor and Downey as presently used to identify low glucosinolate rapeseed is simple but somewhat "messy" and slower than desired. It would be difficult to carry out in many field situations. To date use of this assay (McGregor and Downey) has been limited to analytical labs and crushing plants where facilities for handling and disposing of the activated carbon and for cleaning mortar and pestle, etc. are available.
~ or other analyses, various test devices, laminates, sticks, etc. have been developed to simplify and speed up the procedure. Typical prior art devices are described in United States Patents: 3,011,874; 3,993,451;
4,061,468; 4,065,263; and 4,094,647. To applicant's knowledge, no such devices have dealt with the problem of inhibitors in a colorimetric glucose (or glucosinolate) assay.
Summar~ of the Invention The present invention is primarily an analytical test apparatus suitable for the colorimetric assay of glucose in a liquid composition which also contains inhibitors of the colour development, comprising:
(i) a light-coloured inert solid backing having a sample-` receiving zone (a) spaced apart from an adsorbent zone (b) and test zone (c), (ii) a wick able to transport the liquid by capillary action, ~ .
extending from zone (a) to zone (b) on said backing, .
(iii) a selected adsorbent at zone (b) in capillary contact with the wick, and .. ~ . . . .
: , .. . .
. :
: : . : .
(iv) glucose test paper at test zone (c) on said backing in capillary contact with said adsorbent at a location remote from the wick contact;
the adsorbent being selected to adsorb said inhibitors in preference to glucose; the amount of adsorbent traversed between the wick and the test paper being just sufficient to adsorb the substances which inhibit colour development due to glucose. The test apparatus is most suitably in the form of a stick, strip, tape or sheet.
The invention includes a kit for the colori metric assay of glucose in a composition derived frorn seeds which contain inhibitors of the colour development, comprising:
~a) the test apparatus including appropriate colour standards for the glucose test tape present, (b) means to contain seed sample during crushing, and (c) means to measure and contain both seed sample and water.
T~e kit optionally may include low~ and/or high-glucosinolate seed standards, myrosinase for glucosinolate hydrolysis, or ; seed crushing means.
Description of Drawing The single drawing is a front and side view of a preferred test stick as described in de~ail in the example below.
Detailed Description ` The backing for the test apparatus can be chosen from inter alia sheet, film, stick or tube of plastics, :, ~
; coated cellulosics (wood, paper, etc) or metal foils. The backing should be inert, substantially non-absorbent and of a light colour not interfering with the colour reading at least in the test zone. One backing found very convenient is a photograph mounting sheet having sticky adhesive layers covered on both surfaces by removable paper sheets.
The wick can be made of various porous absorbent papers or fabrics able to conduct liquid by capillary action. Cellulosic wicks in paper form are usually inexpensive and readily available. However, other natural or synthetic fibrous or particulate materials, or sponge material could be used to form the wick.
- The adsorbent can be any high surface area material preferentially a~le to adsorb and hold up the interfering substance or substances. Activated carbon has been found very suitable. Glucose itself will be slowly adsorbed on activated carbon so that the amount of carbon and the time of exposure should be limited to avoid readings which are too low. It was found important in developing a reasonably accurate test apparatus to determine the amount of liquid that would soak up into the glucose test paper and then incorporate into the apparatus upstream of the test paper an amount of adsorbent to give an approximate ratio of : .
; 20 adsorbent to liquid found necessary for an accurate assay.
This ratio of adsorbent to liquid (where the li~uid is about 0.02 to 0.2% glucose) has been determined to be about 1 mg : .
carbon per 17.8 microliters or about 0.6 - 0.7 g per 12.5 ml.
It has been found convenient to form a layer of adsorbent on a thin support, the thickness of this layex being controlled to give the desired amount of adsorbent over an area the size of the adsorbent zone. The support can be adhered readily to the backing in the appropriate location.
A permeable binder may be added to the adsorbent to aid in forming the layer just mentioned. A suitable ~6-:
:`
. . .
. :., , . :
: . .
6''9 binder is gypsum (plaster of paris). Other additives such as chromatographic substrate powder may be added to the adsorbent to aid in capillary transport or selective adsorption in the layer.
The glucose test paper can be any available for glucose determinations, having a good colour response for amounts of glucose ranging from 0.02 to 0.2% wt./vol~ The absorbent action or capillary action of the test paper should be good in order to draw sufficient liquid composition for an accurate determination. A suitable test paper would be that impregnated with glucose oxidase and peroxidase, a chromogen such as o-tolidine and a yellow dye such as F.D.C. yellow No. 5.
The adsorbent layer is delicate and preferably should be protected by an impervious covering such as a I plastic film or tape. This protective covering desirably will extend upstream to cover the wick between zones (a) and (b), in which case the edge of the covering will serve to define the limit of the sample-receiving zone (a). A pro- `
, ~ 20 tective covering may also be applied downstream of the test ~
~ , .
zone (c) to help delineate this zone, and if wick material extends beyond the test paper - to protect this wick. Black plastic tape has been found very suitable as a protective ;: :
~, impervious covering, with strong contrast to the test paper, wick, and backing.
.~
Example A prototype in the form of an analysis stick was constructed using photo print mounting backing consisting of a white sticky center (see drawing at a) which was covered .~, .~. : , ~
with two adhering layers of brown paper (b and h). The brown paper was easily removed to expose a sticky surface.
Only one stic~y surface was used to construct the stick, the other being le~t covered with brown paper. (If desired, the test stick could be built on both sides to give a second or duplicate reading.) It was found convenient to construct 28 analytical sticks, 2.75 inches long by 0.25 inch wide on a 2.75 inch by 7 inch sheet of backing.
To adhere the adsorbent at zone (b), one of the brown paper layers was scored 0.75 inch from one long edge and the 0.75 inch x 7 inch strip of brown paper removed to expose the white sticky center.
To prepare the adsorbent for zone (b), 1 part by wt. of activated carbon powder was mixed with 2 parts by wt. cellulose powder ~grade for thin layer chromatography), 1 part by wt. plaster of paris and enough water to form a slurry. This mixture was spread as a 0.5 mm layer on the back of a glossy medium weight polyethylene-coated photo-graphic enlarging paper which had been fixed and washed.
- 20 This adsorbent layer was allowed to dry overnight and cut into 0.125 x 8.5 inch strips. One of these strips was adhered carbon layer up on the sticky backing next to the :.
brown paper edge as shown in the drawing at c (enlarging ~`
i~ paper) and d (carbon layer). In this way, the amount of ; adsorbent applied in zone (b) was approximately 0.4 mg. (In use when this adsorbent is contacted by about 7 microliters of liquid extract moving through by capillary action, an -approximate ratio would be attained of 1 mg carbon per 17.8 microliters of liquid extract, or 0.7 g carbon per 12.5 ml liquid extract or per g of seed, which previously was found ;~, 8 .
~ .
. . .
32~
to adsorb the inhibitor without appreciably adsorbing the glucose.) The remaining area of the exposed sticky surface was then covered by Whatman No. 1 filter paper as wick, with a slight (1/32 inch) overlap of the adsorbent to provide contact for capillary action (see drawing e).
The 2.125 x 7 inches of brown paper remainin~
was pulled off and a strip of glucose test paper ~Tes-Tape {trademark} Eli Lilly Co.) 3/16 x 7 inches, was applied with a slight overlap (1/32 inch) of the adsorbent to provide for capillary action (see drawing f). Next a 1/16 x 7 inch strip of the same filter paper was applied with a slight overlap of the downstream edge of the glucose test paper, to allow capillary action to continue to beyond the glucose test paper and provide for uniform colour development in the test paper (see drawing g). Because the blue o-tolidine complex ,~ is water-soluble and moves with the capillary action, the width of this final wick was limited to about 1/16 inch, :~
minimizing elution of the colour complex from the test paper.
This final downstream wick is optional but preferred. Instead of being normal wick material, this final strip could be blue litmus paper ~or similar indicator paper) which would turn ., .
~ pink when contacted by the aqueous extract which is acidic ",!~ (pH about 5 - 5.5). This colour change is useful in showing ., .
when capillary action is complete, and may also serve to indicate that the stick has been used for a test in which negligible colour development occurred.
: -The remainder of the exposed white backing was covered with a non-absorbent paper (see drawing h) to provide for handling and labelling. One protective covering of black ~3Z~o~9 plastic non-absorbent tape (see drawing at i) was placed over the adsorbent layer overlapping the glucose test paper slightly, and extending a short distance (0.25 inch) over the wick (e). This tape protects the fragile adsorbent layer and also demarks the level to which this analysis stick is to be submerged in the aqueous extract. By not submerging this taped area or above, the aqueous extract is allowed to move by capillary action up about 0.25 inch of filter paper and through the adsorbent layer before entering the glucose test paper. A
second covering of black plastic non-absorbent tape 0.25 x 7 inches was placed above the glucose test paper (see drawing at j) to contrast and demark the colour development zone (c) and facilitate the colour reading.
Upon completion of this assembly the sheet was cut into 28 analysis sticks of 0.25 x 2.75 inches. These sticks were used to determine glucosinolate content after hydrolysis to glucose, in various oilseeds. A total of 12.5 ml of water was added per g of crushed seed to form the ` aqueous extract and each stick submerged at the wick end up to the edge of the black tape. Full chemical determinations were run on the same extracts using the thiourea method of L.R. Wetter, which involves conversion of isothiocyanates and oxazolidinethiones to their thiourea derivatives in ammonia ethanol. The W absorption of these derivatives was measured at 245 nm and converted to mg equivalents of 3-butenyl isothiocyanate per ~m of oil-free meal. Results are summarized in the following Table.
; 30 ~10-. .
~ .
:- ~ . . .. :
z~
Test-Stick rating Gluco-sinolate Avg~ Range conten~
Brassica napus Tanka 6.2 5-7 17-3 Midas 5.8 5-7 17.2 Golden 6 . o 5-7 lG . o Turret 5-8 5-7 ~5-4 Zephyr 5~Q 4-6 1)~
Target 6.2 5-7 1~.2 Nugget 5-6 5-7 14.1 Oro 4.8 4-5 14.0 Tower 1.2 1-2 2.4 S7N71-1788 1.0 1 1.6 Bronowski 1.0 1 1~3 Brassica campestris R-500 6.6 6-7 14.7 Echo 3-6 ~002 Polar 5.0 4-6 10.1 Span 4.8 4-6 9.
Torch 5- 5 9-~
Arlo 4.6 4-5 8.6 CZY3-1820 1.0 1 1.
~ .
Brassica juncea .
Stoke 6.2 5-7 16.6 Lethbridge 22A 5.2 4-6 14.
Ekla 6.8 6-7 1l~.~
Commercial Brown 6.0 5-7 12.V
Summar~ of the Invention The present invention is primarily an analytical test apparatus suitable for the colorimetric assay of glucose in a liquid composition which also contains inhibitors of the colour development, comprising:
(i) a light-coloured inert solid backing having a sample-` receiving zone (a) spaced apart from an adsorbent zone (b) and test zone (c), (ii) a wick able to transport the liquid by capillary action, ~ .
extending from zone (a) to zone (b) on said backing, .
(iii) a selected adsorbent at zone (b) in capillary contact with the wick, and .. ~ . . . .
: , .. . .
. :
: : . : .
(iv) glucose test paper at test zone (c) on said backing in capillary contact with said adsorbent at a location remote from the wick contact;
the adsorbent being selected to adsorb said inhibitors in preference to glucose; the amount of adsorbent traversed between the wick and the test paper being just sufficient to adsorb the substances which inhibit colour development due to glucose. The test apparatus is most suitably in the form of a stick, strip, tape or sheet.
The invention includes a kit for the colori metric assay of glucose in a composition derived frorn seeds which contain inhibitors of the colour development, comprising:
~a) the test apparatus including appropriate colour standards for the glucose test tape present, (b) means to contain seed sample during crushing, and (c) means to measure and contain both seed sample and water.
T~e kit optionally may include low~ and/or high-glucosinolate seed standards, myrosinase for glucosinolate hydrolysis, or ; seed crushing means.
Description of Drawing The single drawing is a front and side view of a preferred test stick as described in de~ail in the example below.
Detailed Description ` The backing for the test apparatus can be chosen from inter alia sheet, film, stick or tube of plastics, :, ~
; coated cellulosics (wood, paper, etc) or metal foils. The backing should be inert, substantially non-absorbent and of a light colour not interfering with the colour reading at least in the test zone. One backing found very convenient is a photograph mounting sheet having sticky adhesive layers covered on both surfaces by removable paper sheets.
The wick can be made of various porous absorbent papers or fabrics able to conduct liquid by capillary action. Cellulosic wicks in paper form are usually inexpensive and readily available. However, other natural or synthetic fibrous or particulate materials, or sponge material could be used to form the wick.
- The adsorbent can be any high surface area material preferentially a~le to adsorb and hold up the interfering substance or substances. Activated carbon has been found very suitable. Glucose itself will be slowly adsorbed on activated carbon so that the amount of carbon and the time of exposure should be limited to avoid readings which are too low. It was found important in developing a reasonably accurate test apparatus to determine the amount of liquid that would soak up into the glucose test paper and then incorporate into the apparatus upstream of the test paper an amount of adsorbent to give an approximate ratio of : .
; 20 adsorbent to liquid found necessary for an accurate assay.
This ratio of adsorbent to liquid (where the li~uid is about 0.02 to 0.2% glucose) has been determined to be about 1 mg : .
carbon per 17.8 microliters or about 0.6 - 0.7 g per 12.5 ml.
It has been found convenient to form a layer of adsorbent on a thin support, the thickness of this layex being controlled to give the desired amount of adsorbent over an area the size of the adsorbent zone. The support can be adhered readily to the backing in the appropriate location.
A permeable binder may be added to the adsorbent to aid in forming the layer just mentioned. A suitable ~6-:
:`
. . .
. :., , . :
: . .
6''9 binder is gypsum (plaster of paris). Other additives such as chromatographic substrate powder may be added to the adsorbent to aid in capillary transport or selective adsorption in the layer.
The glucose test paper can be any available for glucose determinations, having a good colour response for amounts of glucose ranging from 0.02 to 0.2% wt./vol~ The absorbent action or capillary action of the test paper should be good in order to draw sufficient liquid composition for an accurate determination. A suitable test paper would be that impregnated with glucose oxidase and peroxidase, a chromogen such as o-tolidine and a yellow dye such as F.D.C. yellow No. 5.
The adsorbent layer is delicate and preferably should be protected by an impervious covering such as a I plastic film or tape. This protective covering desirably will extend upstream to cover the wick between zones (a) and (b), in which case the edge of the covering will serve to define the limit of the sample-receiving zone (a). A pro- `
, ~ 20 tective covering may also be applied downstream of the test ~
~ , .
zone (c) to help delineate this zone, and if wick material extends beyond the test paper - to protect this wick. Black plastic tape has been found very suitable as a protective ;: :
~, impervious covering, with strong contrast to the test paper, wick, and backing.
.~
Example A prototype in the form of an analysis stick was constructed using photo print mounting backing consisting of a white sticky center (see drawing at a) which was covered .~, .~. : , ~
with two adhering layers of brown paper (b and h). The brown paper was easily removed to expose a sticky surface.
Only one stic~y surface was used to construct the stick, the other being le~t covered with brown paper. (If desired, the test stick could be built on both sides to give a second or duplicate reading.) It was found convenient to construct 28 analytical sticks, 2.75 inches long by 0.25 inch wide on a 2.75 inch by 7 inch sheet of backing.
To adhere the adsorbent at zone (b), one of the brown paper layers was scored 0.75 inch from one long edge and the 0.75 inch x 7 inch strip of brown paper removed to expose the white sticky center.
To prepare the adsorbent for zone (b), 1 part by wt. of activated carbon powder was mixed with 2 parts by wt. cellulose powder ~grade for thin layer chromatography), 1 part by wt. plaster of paris and enough water to form a slurry. This mixture was spread as a 0.5 mm layer on the back of a glossy medium weight polyethylene-coated photo-graphic enlarging paper which had been fixed and washed.
- 20 This adsorbent layer was allowed to dry overnight and cut into 0.125 x 8.5 inch strips. One of these strips was adhered carbon layer up on the sticky backing next to the :.
brown paper edge as shown in the drawing at c (enlarging ~`
i~ paper) and d (carbon layer). In this way, the amount of ; adsorbent applied in zone (b) was approximately 0.4 mg. (In use when this adsorbent is contacted by about 7 microliters of liquid extract moving through by capillary action, an -approximate ratio would be attained of 1 mg carbon per 17.8 microliters of liquid extract, or 0.7 g carbon per 12.5 ml liquid extract or per g of seed, which previously was found ;~, 8 .
~ .
. . .
32~
to adsorb the inhibitor without appreciably adsorbing the glucose.) The remaining area of the exposed sticky surface was then covered by Whatman No. 1 filter paper as wick, with a slight (1/32 inch) overlap of the adsorbent to provide contact for capillary action (see drawing e).
The 2.125 x 7 inches of brown paper remainin~
was pulled off and a strip of glucose test paper ~Tes-Tape {trademark} Eli Lilly Co.) 3/16 x 7 inches, was applied with a slight overlap (1/32 inch) of the adsorbent to provide for capillary action (see drawing f). Next a 1/16 x 7 inch strip of the same filter paper was applied with a slight overlap of the downstream edge of the glucose test paper, to allow capillary action to continue to beyond the glucose test paper and provide for uniform colour development in the test paper (see drawing g). Because the blue o-tolidine complex ,~ is water-soluble and moves with the capillary action, the width of this final wick was limited to about 1/16 inch, :~
minimizing elution of the colour complex from the test paper.
This final downstream wick is optional but preferred. Instead of being normal wick material, this final strip could be blue litmus paper ~or similar indicator paper) which would turn ., .
~ pink when contacted by the aqueous extract which is acidic ",!~ (pH about 5 - 5.5). This colour change is useful in showing ., .
when capillary action is complete, and may also serve to indicate that the stick has been used for a test in which negligible colour development occurred.
: -The remainder of the exposed white backing was covered with a non-absorbent paper (see drawing h) to provide for handling and labelling. One protective covering of black ~3Z~o~9 plastic non-absorbent tape (see drawing at i) was placed over the adsorbent layer overlapping the glucose test paper slightly, and extending a short distance (0.25 inch) over the wick (e). This tape protects the fragile adsorbent layer and also demarks the level to which this analysis stick is to be submerged in the aqueous extract. By not submerging this taped area or above, the aqueous extract is allowed to move by capillary action up about 0.25 inch of filter paper and through the adsorbent layer before entering the glucose test paper. A
second covering of black plastic non-absorbent tape 0.25 x 7 inches was placed above the glucose test paper (see drawing at j) to contrast and demark the colour development zone (c) and facilitate the colour reading.
Upon completion of this assembly the sheet was cut into 28 analysis sticks of 0.25 x 2.75 inches. These sticks were used to determine glucosinolate content after hydrolysis to glucose, in various oilseeds. A total of 12.5 ml of water was added per g of crushed seed to form the ` aqueous extract and each stick submerged at the wick end up to the edge of the black tape. Full chemical determinations were run on the same extracts using the thiourea method of L.R. Wetter, which involves conversion of isothiocyanates and oxazolidinethiones to their thiourea derivatives in ammonia ethanol. The W absorption of these derivatives was measured at 245 nm and converted to mg equivalents of 3-butenyl isothiocyanate per ~m of oil-free meal. Results are summarized in the following Table.
; 30 ~10-. .
~ .
:- ~ . . .. :
z~
Test-Stick rating Gluco-sinolate Avg~ Range conten~
Brassica napus Tanka 6.2 5-7 17-3 Midas 5.8 5-7 17.2 Golden 6 . o 5-7 lG . o Turret 5-8 5-7 ~5-4 Zephyr 5~Q 4-6 1)~
Target 6.2 5-7 1~.2 Nugget 5-6 5-7 14.1 Oro 4.8 4-5 14.0 Tower 1.2 1-2 2.4 S7N71-1788 1.0 1 1.6 Bronowski 1.0 1 1~3 Brassica campestris R-500 6.6 6-7 14.7 Echo 3-6 ~002 Polar 5.0 4-6 10.1 Span 4.8 4-6 9.
Torch 5- 5 9-~
Arlo 4.6 4-5 8.6 CZY3-1820 1.0 1 1.
~ .
Brassica juncea .
Stoke 6.2 5-7 16.6 Lethbridge 22A 5.2 4-6 14.
Ekla 6.8 6-7 1l~.~
Commercial Brown 6.0 5-7 12.V
5% Midas in Tower* 1.4 1-2 ~-3 10% hlidas in Tower 1.6 1-3 3-3 15% Midas in Tower 2.2 2-3 3.7 20% rlidas in Tower 3.4 3-4 4.3 40% Midas in Tower 4.2 3-5 7-3 +Average of 5 determinations rated on the 0-9 rating scale, O repre-senting no color change, 2, 4, 6 and 8 corresponding to the color standards on the Tes-Tape pack~ge.
~Avera~e of 2 determinations by the thiourea method expressed as mg equivalents of 3-butenyl isothiocyanate per gram of oil-free meal.
*Admixture by weight.
.
.
.
2~
The test stick method was consistently able to identify low glucosinolate cultivars. Tests with admixtures of seeds of high and low glucosinolate content confirmed that the test stick method could consistently detect contamination of low glucosinolate seed by high glucosinolate seed when the overall glucosinolate was raised to 3 mg or more expressed as equivalents of 3-butenyl isothiocyanate per g of oil-free meal.
The test apparatus is thus very useful for identifying seed of low glucosinolate cultivars throughout the transportation and marketing system. It is anticipated that the test apparatus will be used by farmers to verify that the seed they are sowing is low in glucosinolate content, by truckers to verify that the seed they are hauling is low in glucosinolate content, by crushing plants and country elevators to identify deliveries of low glucosinolate seed, and generally throughout the processing and marketing system to segregate seed of high and low glucosinolate content.
Thus many tests will be done under field conditions and it would be desirable to have a kit including the test apparatus to facilitate the entire assay.
A kit has been designed which cPntainS
; disposable plastic containers able to withstand crushing of the seed contained therein and within which water can be admixed with the crushed seed. Separate measuring containers or other means for measuring the seed sample and water are also provided. Optionally, the kit can include a sample o known low-glucosinolate content seed which can serve as a standard for comparison, and also as a source of myrosinase where myrosinase-free non-hydrolyzed glucosinolate compositions are to be assayed. For instance, for analysis of commercial z ~
rapeseed meal a source of myrosinase must be added since the seed-borne enzyme activity is destroyed by commerical oil extraction processing. Alternatively, myrosinase itself or another source thereof (such as an oil-free non-heated low-glucosinolate meal) can be present. For convenience, in kits intended for use with commercial meals or other non-hydro-lyzed, myrosinase-free samples only, the source of myrosinase could be placed in the plastic container to be used for crushing, kneading, mixing, etc. The kit may also contain seed crushing means able to exert sufficient compression or impact to crush the seed (e.g. hammer or screw compression means). The test apparatus, test procedure, and colour standards for the glucose test tape would always be included.
With such a kit the assay would be performed as follows.
A small graduated container would be filled to the mark with seed (e.g. 1 g) and the seed transferred to a sturdy plastic bag (suitably having a zipper-type closure).
The seed in the closed bag would be crushed, e.g. with hammer impacts, taking care not to rupture the bag. A
measured amount of water (e.g. ~.5 ml) would be transferred to the bag and the crushed seed and water kneaded to form a paste, kneading or mixing continuing for about 2 min. to allow for enzyme action on the glucosinolates. A further measured amount of water (e.g. 10 ml) would then be added to the bag and the contents mixed to form an extract. Only the wick portion (zone (a) ) of the test apparatus would be contacted up to the mark thereon. When the extract has moved up through the wick, adsorbent and glucose test paper, the test apparatus is separated from the extract and let stand for about 3 min. Green colour development in the yellow Z~
glucose test paper may be rated by comparison to colour produced by seed samples of known glucosinolate content or by comparison with the colour standards provided. On a rating scale of 0 to 9 where 0 represents no colour change and 2, 4, 6 and 8 correspond to the colour standards provided, low glucosinolate seed should rate no higher than 3. The plastic bag and ana~ysis test apparatus are discarded after use.
As described in the Example above, one very convenient and compact analysis stick would have the approxi-mate dimensions measured consecutively in the downstream . direction starting from one end:
:: sample-receiving zone (a) 12.7 mm wick length between zones (a) and (b) 3.2 mm i total wick length 15.9 mm ~ adsorbent zone (b) 3.2 mm .. .
test zone (c) 4.8 mm with the width being approximately 6.4 mm and the weight of carbon adsorbent being about 0.4 mg. This size permits use . ~ 20 of the standard width of the Tes-Tape (3/16 inch or about ~
.~.
!~' 4.8 mm) for the extent of zone (c).
.
",~ .
.
~' . ' .
~Avera~e of 2 determinations by the thiourea method expressed as mg equivalents of 3-butenyl isothiocyanate per gram of oil-free meal.
*Admixture by weight.
.
.
.
2~
The test stick method was consistently able to identify low glucosinolate cultivars. Tests with admixtures of seeds of high and low glucosinolate content confirmed that the test stick method could consistently detect contamination of low glucosinolate seed by high glucosinolate seed when the overall glucosinolate was raised to 3 mg or more expressed as equivalents of 3-butenyl isothiocyanate per g of oil-free meal.
The test apparatus is thus very useful for identifying seed of low glucosinolate cultivars throughout the transportation and marketing system. It is anticipated that the test apparatus will be used by farmers to verify that the seed they are sowing is low in glucosinolate content, by truckers to verify that the seed they are hauling is low in glucosinolate content, by crushing plants and country elevators to identify deliveries of low glucosinolate seed, and generally throughout the processing and marketing system to segregate seed of high and low glucosinolate content.
Thus many tests will be done under field conditions and it would be desirable to have a kit including the test apparatus to facilitate the entire assay.
A kit has been designed which cPntainS
; disposable plastic containers able to withstand crushing of the seed contained therein and within which water can be admixed with the crushed seed. Separate measuring containers or other means for measuring the seed sample and water are also provided. Optionally, the kit can include a sample o known low-glucosinolate content seed which can serve as a standard for comparison, and also as a source of myrosinase where myrosinase-free non-hydrolyzed glucosinolate compositions are to be assayed. For instance, for analysis of commercial z ~
rapeseed meal a source of myrosinase must be added since the seed-borne enzyme activity is destroyed by commerical oil extraction processing. Alternatively, myrosinase itself or another source thereof (such as an oil-free non-heated low-glucosinolate meal) can be present. For convenience, in kits intended for use with commercial meals or other non-hydro-lyzed, myrosinase-free samples only, the source of myrosinase could be placed in the plastic container to be used for crushing, kneading, mixing, etc. The kit may also contain seed crushing means able to exert sufficient compression or impact to crush the seed (e.g. hammer or screw compression means). The test apparatus, test procedure, and colour standards for the glucose test tape would always be included.
With such a kit the assay would be performed as follows.
A small graduated container would be filled to the mark with seed (e.g. 1 g) and the seed transferred to a sturdy plastic bag (suitably having a zipper-type closure).
The seed in the closed bag would be crushed, e.g. with hammer impacts, taking care not to rupture the bag. A
measured amount of water (e.g. ~.5 ml) would be transferred to the bag and the crushed seed and water kneaded to form a paste, kneading or mixing continuing for about 2 min. to allow for enzyme action on the glucosinolates. A further measured amount of water (e.g. 10 ml) would then be added to the bag and the contents mixed to form an extract. Only the wick portion (zone (a) ) of the test apparatus would be contacted up to the mark thereon. When the extract has moved up through the wick, adsorbent and glucose test paper, the test apparatus is separated from the extract and let stand for about 3 min. Green colour development in the yellow Z~
glucose test paper may be rated by comparison to colour produced by seed samples of known glucosinolate content or by comparison with the colour standards provided. On a rating scale of 0 to 9 where 0 represents no colour change and 2, 4, 6 and 8 correspond to the colour standards provided, low glucosinolate seed should rate no higher than 3. The plastic bag and ana~ysis test apparatus are discarded after use.
As described in the Example above, one very convenient and compact analysis stick would have the approxi-mate dimensions measured consecutively in the downstream . direction starting from one end:
:: sample-receiving zone (a) 12.7 mm wick length between zones (a) and (b) 3.2 mm i total wick length 15.9 mm ~ adsorbent zone (b) 3.2 mm .. .
test zone (c) 4.8 mm with the width being approximately 6.4 mm and the weight of carbon adsorbent being about 0.4 mg. This size permits use . ~ 20 of the standard width of the Tes-Tape (3/16 inch or about ~
.~.
!~' 4.8 mm) for the extent of zone (c).
.
",~ .
.
~' . ' .
Claims (18)
1. An analytical test apparatus suitable for the colorimetric assay of glucose in a liquid composition which also contains inhibitors of the colour development, comprising:
(i) a light-coloured inert solid backing having a sample-receiving zone (a) spaced apart from an adsorbent zone (b) and test zone (c), (ii) a wick able to transport the liquid by capillary action, extending from zone (a) to zone (b) on said backing, (iii) a selected adsorbent at zone (b) in capillary contact with the wick, and (iv) glucose test paper at test zone (c) on said backing in capillary contact with said adsorbent at a location remote from the wick contact;
the adsorbent being selected to adsorb said inhibitors in preference to glucose; the amount of adsorbent traversed between the wick and the test paper being just sufficient to adsorb the substances which inhibit colour development due to glucose.
(i) a light-coloured inert solid backing having a sample-receiving zone (a) spaced apart from an adsorbent zone (b) and test zone (c), (ii) a wick able to transport the liquid by capillary action, extending from zone (a) to zone (b) on said backing, (iii) a selected adsorbent at zone (b) in capillary contact with the wick, and (iv) glucose test paper at test zone (c) on said backing in capillary contact with said adsorbent at a location remote from the wick contact;
the adsorbent being selected to adsorb said inhibitors in preference to glucose; the amount of adsorbent traversed between the wick and the test paper being just sufficient to adsorb the substances which inhibit colour development due to glucose.
2. The test apparatus of claim 1 in the form of a stick, strip, tape or sheet.
3. The test apparatus of claim 1 adapted for the rapid determination of hydrolyzed glucosinolates in oilseeds or oilseed meal, and wherein the adsorbent is activated carbon.
4. The test apparatus of claims 1, 2 or 3 wherein the backing is a white plastic.
5. The test apparatus of claims 1, 2 or 3 wherein the wick material is a porous cellulosic paper or fabric.
6. The test apparatus of claim 3 wherein the activated carbon was preformed into a layer with a small amount of permeable binder, and the layer fixed to said backing.
7. The test apparatus of claim 3 wherein the activated carbon adsorbent comprises a fluid-conducting chromatographic substrate.
8. The test apparatus of claims 3, 6 or 7 wherein the activated carbon adsorbent comprises chromato-graphic-grade cellulose powder and gypsum binder.
9. The test apparatus of claims 1, 2 or 3 wherein a narrow band of wick material is in capillary contact with the downstream end of the glucose test paper.
10. The test apparatus of claims 1, 2 or 3 including a protective covering over the adsorbent at zone (b).
11. The test apparatus of claims 1, 2 or 3 including opaque protective coverings both upstream and downstream of the glucose test paper thus outlining the colour change test zone (c).
12. The test apparatus of claims 1, 2 or 3 including means delimiting the sample-receiving zone (a).
13. The test apparatus of claims 1 or 3 in the form of an elongated stick, strip or tape with approximate dimensions measured consecutively in the downstream direction starting from one end:
sample-receiving zone (a) 12.7 mm wick length between zones (a) and (b) 3.2 mm total wick length 15.9 mm adsorbent zone (b) 3.2 mm test zone (c) 4.8 mm with the width being approximately 6.4 mm and the weight of carbon adsorbent being ahout 0.4 mg.
sample-receiving zone (a) 12.7 mm wick length between zones (a) and (b) 3.2 mm total wick length 15.9 mm adsorbent zone (b) 3.2 mm test zone (c) 4.8 mm with the width being approximately 6.4 mm and the weight of carbon adsorbent being ahout 0.4 mg.
14. A kit for the colorimetric assay of glucose in a composition derived from seeds which contain inhibitors of the colour development, comprising:
a) the test apparatus as in claim 1, including appropriate colour standards for the glucose test paper present, (b) means to contain seed sample during crushing, and (c) means to measure and contain both seed sample and water.
a) the test apparatus as in claim 1, including appropriate colour standards for the glucose test paper present, (b) means to contain seed sample during crushing, and (c) means to measure and contain both seed sample and water.
15. The kit of claim 14 including a sample of low-glucosinolate seed serving as a standard.
16. The kit of claim 14 including myrosinase or a source of myrosinase.
17. The kit of claim 14, 15 or 16 wherein the means (b) is a flexible, co}lapsible plastic container able to withstand compression or impacts sufficient to crush the seed.
18. The kit of claims 14, 15 or 16 including seed cxushing means.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA330,984A CA1113279A (en) | 1979-06-19 | 1979-06-19 | Colorimetric analytical test apparatus |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA330,984A CA1113279A (en) | 1979-06-19 | 1979-06-19 | Colorimetric analytical test apparatus |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1113279A true CA1113279A (en) | 1981-12-01 |
Family
ID=4114596
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA330,984A Expired CA1113279A (en) | 1979-06-19 | 1979-06-19 | Colorimetric analytical test apparatus |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA1113279A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102692387A (en) * | 2012-06-15 | 2012-09-26 | 南京农业大学 | Method for determining total content of glucosinolates in plants |
-
1979
- 1979-06-19 CA CA330,984A patent/CA1113279A/en not_active Expired
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102692387A (en) * | 2012-06-15 | 2012-09-26 | 南京农业大学 | Method for determining total content of glucosinolates in plants |
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