WO2001002859A1 - Benzoate buffers for zone electrophoresis and immunofixation - Google Patents
Benzoate buffers for zone electrophoresis and immunofixation Download PDFInfo
- Publication number
- WO2001002859A1 WO2001002859A1 PCT/DK2000/000350 DK0000350W WO0102859A1 WO 2001002859 A1 WO2001002859 A1 WO 2001002859A1 DK 0000350 W DK0000350 W DK 0000350W WO 0102859 A1 WO0102859 A1 WO 0102859A1
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- WIPO (PCT)
- Prior art keywords
- buffer
- salt
- benzoic acid
- concentration
- gel
- Prior art date
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/447—Systems using electrophoresis
- G01N27/44704—Details; Accessories
Definitions
- the present invention relates to buffers comprising benzoic acid and/or a salt thereof for use in zone electrophoresis and/or immunofixation.
- the invention further relates to the use of benzoic acid and/or a salt thereof as buffer components for zone electrophoresis and/or immunofixation.
- the invention also concerns kits for zone electrophoresis and/or immunofixation.
- An optimised combination of the gel buffer and the compartment buffer is disclosed herein giving sharper and easier identifiable protein bands or protein pattern. A special advantage being that the buffers disclosed herein are non-hazardous.
- Immunofixation is a widely used diagnostic method. It is a rapid, important and useful tool for the examination and identification of various protein abnormalities in serum, urine, cerebrospinal and synovial fluids.
- the immunofixation procedure can be used for the identification of any single protein band of an electrophoresis.
- the technique is a combination of zone electrophoresis followed by immunofixation using monospecific antibodies. In this way it is possible to separate and identify different proteins in a biological mixture according to their physicochemical properties and antigenic properties.
- the immunofixation procedure is most frequently used for the detection of monoclonal immunoglobulins in serum and Bence Jones proteins in urine.
- barbital buffers comprising barbituric acid and/or sodium barbiturate are used (ref. 3) .
- this use is recommended as barbituric acid/sodium barbiturate provide a good separation of all protein bands.
- barbituric acid and sodium barbiturate are hazardous compounds which potentially cause irritation by contact with the skin, the eyes or the respiratory system, and which in extreme cases even may cause death. In more and more countries, the use of barbituric acid and sodium barbiturate in buffers is therefore prohibited.
- the present invention provides such compounds which can replace barbituric acid and sodium barbiturate .
- the present invention relates to buffers comprising benzoic acid and/or a salt thereof for zone electrophoresis and/or immunofixation.
- the present invention relates to the use of benzoic acid and/or a salt thereof as buffer components for zone electrophoresis and/or immunofixation.
- the present invention concerns kits for zone electrophoresis and/or immunofixation comprising a buffer as described herein.
- Figure 1 shows an immunofixation gel obtained following the procedures described in Example 1.
- FIGS 2A, 2B, 2C and 2D show four patient serum samples subjected to zone electrophoresis and immunofixation with the buffer of the invention comprising the sodium salt of benzoic acid.
- Figures 3A, 3B, 3C and 3D show the four patient serum samples as shown in Figures 2A, 2B, 2C and 2D subjected to zone electrophoresis and immunofixation with the buffer of the invention comprising the ammonium salt of benzoic acid.
- the present invention relates buffers for use in zone electrophoresis and/or immunofixation comprising benzoic acid and/or a salt thereof.
- a non-barbiturate buffer composition for use in the electrophoretic separation of proteins into fractions comprising a water soluble salicylate such as sodium salicylate and an inorganic salt.
- the salicylate is used in a concentration of from 0.5 to 1 g salicylate to 1.5 to 3 g inorganic salt.
- the buffer of the invention may comprise a salt of benzoic acid.
- suitable salts is the sodium salt, the potassium salt, the calcium salt, the magnesium salt and/or the ammonium salt of benzoic acid.
- the buffer of the invention may comprise one or more of these salts of benzoic acid optionally in combination with benzoic acid itself. Accordingly, benzoic acid or one or more salts of benzoic acid may be used alone, or benzoic acid and one or more salts of benzoic acid may be used in combination.
- the buffer of the invention comprises a salt of benzoic acid, in particular the sodium salt of benzoic acid.
- the buffer of the invention is particularly suitable for separation of proteins, serum proteins, and im unoglobulins in zone electrophoresis and/or immunofixation.
- Proteins are large molecules which are susceptible to denaturation, thus, making them sensitive to the conditions employed in the separation procedure. E.g. heat development during the electrophoresis procedure may result m destruction of the protein structure.
- Increased ionic strength of the running buffer increases the electric conductivity, thus leading to an increased heat development. This can be avoided e.g. by using heavy cooling procedures during the electrophoresis procedure.
- low concentration of lon- providmg components of the running buffer can be used. For instance, m US 4,321,119, a relatively low concentration of salicylate is used.
- a buffer comprising benzoic acid and/or a salt thereof does not suffer from the above drawbacks.
- the benzoic acid and the salts thereof can be used m relatively high concentrations without the need for additional cooling m order to avoid breakdown of the protein structure or denaturation.
- An additional advantageous feature of the buffer is that the benzoic acid and the salts thereof m general act as preserving agents. This is especially observed m the case of sodium benzoate (the sodium salt of benzoic acid) .
- the buffer of the invention is used as a gel buffer and/or as a compartment buffer.
- the term ' "buffer" includes both.
- the gel buffer and the compartment buffer may have the same acid/salt concentration, or the concentrations of the gel buffer and the compartment buffer may be different.
- the gel buffer suitably comprises benzoic acid and/or a salt thereof m a concentration of from 1 to 10 g/L.
- the buffer comprises benzoic acid and/or a salt thereof in a concentration of from 3 to 8 g/L, 3.5 to 8 g/L, 4 to 8 g/L, 4.5 to 8 g/L, 5 to 8 g/L, 5.5 to 8 g/L, 6 to 8 g/L, 6.5 to 8 g/L, 7 to 8 g/L, or 7.5 to 8 g/L.
- the concentration of benzoic acid and/or the salt thereof may be 7 g/L. Surprisingly, it seems as if the concentration range yielding a good separation is peak-like, having a maximum in the concentration range from 6.5 to 7.5 g/L, more specifically around a concentration of about 7 g/L.
- the compartment buffer suitably comprises benzoic acid and/or a salt thereof in a concentration of from 1 to 10 g/L.
- the buffer comprises benzoic acid and/or a salt thereof in a concentration of from 1 to 5 g/L, 1.5 to 5 g/L, 2 to 5 g/L, 2.5 to 5 g/L, 3 to 5 g/L, 3.5 to 5 g/L, 4 to 5 g/L, or 4.5 to 5 g/L.
- the concentration of benzoic acid and/or the salt thereof may be 3.5 g/L. Surprisingly, it seems as if the concentration range yielding a good separation is peak-like, having a maximum in the concentration range 3 to 4 g/L, more specifically around a concentration of about 3.5 g/L.
- the zone electrophoresis/immunofixation procedure is a very powerful tool in the early diagnosis of various diseases. Frequently, the disease can be diagnosed even before the patient experiences symptoms of the disease.
- the buffers of the invention provide such good and distinct separation enabling a reliable diagnosis.
- the buffer of the invention may further comprise additional components such as Tris and/or Tricine and/or calcium lactate and/or sodium azide.
- the benzoic acid compounds as defined above are suitable as buffer components, in particular as gel buffers and compartment buffers. They are much less hazardous than the conventionally used barbiturates. Furthermore, no hazard labelling of the buffers is required. It has further been shown (cf. Example 1) that the buffers of the invention seem to provide sharper and more well defined bands than the conventionally used barbiturate- containing buffers. Also, the use of a compartment buffer having a lower salt/acid concentration than the gel buffer may be advantageous. This may inhibit the heat development during electrophoresis and yield sharper and more well defined bands.
- the buffer may contain one or more additional components such as buffering agents, preserving agents, colouring agents, salts, detergent and surfactants .
- the gel buffer comprises the sodium salt of benzoic acid in a concentration of from 3 to 8 g/L, 3.5 to 8 g/L, 4 to 8 g/L, 4.5 to 8 g/L, 5 to 8 g/L, 5.5 to 8 g/L, 6 to 8 g/L, 6.5 to 8 g/L, 7 to 8 g/L, or 7.5 to 8 g/L.
- the concentration of the sodium salt of benzoic acid is from 6.5 to 7.5 g/L, in particular 7 g/L.
- the compartment buffer comprises the sodium salt of benzoic acid in a concentration of from 1 to 5 g/L, 1.5 to 5 g/L, 2 to 5 g/L, 2.5 to 5 g/L, 3 to 5 g/L, 3.5 to 5 g/L, 4 to 5 g/L, or 4.5 to 5 g/L.
- the concentration of the sodium salt of benzoic acid is from 3 to 4 g/L, in particular 3.5 g/L.
- the gel buffer may have a salt concentration of 7 g/L, and the compartment buffer a concentration of 3.5 g/L.
- the buffer is for use in zone electrophoresis, and/or immunofixation.
- the test samples are suitably serum, urine, cerebrospinal or synovial fluids .
- the present invention relates to the use of benzoic acid and/or a salt thereof as a buffer component for zone electrophoresis and/or immunofixation.
- Benzoic acid or a salt of benzoic acid may be used alone, or benzoic acid and one or more salts of benzoic acid may be used in combination.
- suitable salts of benzoic acid are the sodium salt, the potassium salt, the calcium salt, the magnesium salt and the ammonium salt.
- the salt of benzoic acid is the sodium salt.
- the present invention relates to the use of benzoic acid and/or a salt thereof, wherein the benzoic acid and/or the salt thereof is present in a concentration of from 1 to 10 g/L.
- benzoic acid and/or the salt thereof may be used in a concentration of from 3 to 8 g/L, 3.5 to 8 g/L, 4 to 8 g/L, 4.5 to 8 g/L, 5 to 8 g/L, 5.5 to 8 g/L, 6 to 8 g/L, 6.5 to 8 g/L, 7 to 8 g/L, or 7.5 to 8 g/L.
- the concentration may be from 6.5 to 7.5 g/L, like 7 g/L.
- benzoic acid and/or the salt thereof may be used in a concentration of from 1 to 5 g/L, 1.5 to 5 g/L, 2 to 5 g/L, 2.5 to 5 g/L, 3 to 5 g/L, 3.5 to 5 g/L, 4 to 5 g/L, or 4.5 to 5 g/L.
- the concentration may be from 3 to 4 g/L, like 3.5 g/L.
- the sodium salt of benzoic acid in a concentration of from 3 to 8 g/L, 3.5 to 8 g/L,
- the sodium salt of benzoic acid in a concentration of from 6.5 to 7.5 g/L, in particular a concentration of 7 g/L, is used as a component of the gel buffer.
- the sodium salt of benzoic acid in a concentration of from 1 to 5 g/L, 1.5 to 5 g/L,
- the sodium salt of benzoic acid in a concentration of from 3 to 4 g/L, in particular a concentration of 3.5 g/L, is used as a component of the compartment buffer.
- the present invention relates to the use of benzoic acid and/or a salt thereof in a buffer for electrophoresis and/or immunofixation, wherein the buffer further comprises Tris and/or Tricine and/or calcium lactate and/or sodium azide.
- kits for zone electrophoresis and/or immunofixation which kits comprise a buffer as defined above.
- the kit further comprises gels containing the buffer of the invention, staining solutions, antibodies (e.g. rabbit immunoglobulins), blotters, templates, fixation and/or reagents.
- staining solutions e.g. staining solutions
- antibodies e.g. rabbit immunoglobulins
- blotters e.g. blotters
- templates e.g. fixation and/or reagents.
- the gel to be used is suitably an agarose gel.
- the agarose gel may suitably be provided in a ready-to-use packing containing the buffer of the invention.
- the buffer of the invention may thus be used as compartment buffer as well as gel buffer for supporting medias such as agarose, starch, polyacrylamide etc.
- the invention is further illustrated by the following, non-limiting example.
- the content of each of the bottles of buffer is diluted prior to use to a total volume of 1000 mL with distilled water.
- the diluted buffer contains sodium benzoate (3.5 g/L) or ammonium benzoate (3.5 g/L), Tris (3.6 g/L), Tricine (0.6 g/L), calcium lactate (0.75 g/L), and sodium azide (0.04 g/L) .
- Concentrated Staining Solution 75 mL (4 x concentrated) . Amido Black in 5% acetic acid. The Staining Solution is diluted prior to use to a total volume of 300 mL with distilled water. The concentration of Amido Black in the diluted Solution is 5 g/L.
- Test Sample Serum samples, optionally freshly drawn. 33 samples were tested.
- Antibody Template 10 pieces.
- Sample Blotter Pre-cut disposable, filter paper, 1 package, 10 sheets.
- Drying Blotter Pre-cut disposable filter paper, 2 package, 20 sheets each. Fixation Reagents. Protein Fixative Solution 1.0 mL containing 7% sulphosalicylic acid and 5% acetic acid. Green dyed.
- Electrophoresis apparatus for Agarose Gels (DAKO Electrophoresis Apparatus Code No. S 2200) . Pipettes (5 ⁇ L, 80 ⁇ L) . Containers for washing, staining and destaining of Agarose Gels (DAKO Washing and Staining Accessory Kit Code No. S 2201) . Glass plate (minimum 11x11 cm) plus a weight of approximately 1 kg for pressing the gel. Hair dryer or a drying oven (maximum 90°C) . Additional Reagents. Rabbit Anti-Human IgD (DAKO Code No. A 0093) , specific for ⁇ -chains, immunoglobulin fraction, preserved with 15 mM sodium azide.
- Rabbit Anti-Human IgE (DAKO Code No. A 0094), specific for ⁇ -chains, immunoglobulin fraction, preserved with 15 mM sodium azide.
- Rabbit Anti-Human Kappa Free Light Chains (DAKO Code No. A 0100), specific for kappa free light chains, immunoglobulin fraction, preserved with 15 mM sodium azide.
- Rabbit Anti-Human Lambda Free Light Chains (DAKO Code No. A 0101), specific for lambda free light chains, immunoglobulin fraction, preserved with 15 mM sodium azide.
- All serum specimens should preferably be diluted with saline solution just prior to use.
- serum should be diluted 1:4 (1 part serum + 3 parts Saline Solution).
- serum should be diluted 1:15 (1 part serum + 14 parts saline solution) .
- a dilution of 1:4 is recommended.
- serum specimens suspected of containing high levels of monoclonal immunoglobulin >30 g/L
- a dilution of 1:31 may be suitable.
- the urine sample For the detection of Bence Jones proteins in urine, the urine sample should be concentrated (e.g. by ultrafiltration) to a total protein concentration of at least 1 g/L. This concentrated urine is applied in all slots.
- the light chain antibodies as described above will precipitate kappa or lambda chains whether they are free or still part of the immunoglobulin molecule.
- special antibodies as described above against free kappa and free lambda light chains could be employed.
- Al1 samples are prepared as described above.
- the Agarose Gel is removed from the foil package and placed on a level surface. Excess moisture is removed from the gel surface by gentle blotting with a Gel Blotter.
- the Sample Template is placed on the surface of the gel so that the slots are in alignment with the arrows located on the edges of the gel. 5 ⁇ L of the pre-diluted serum sample is applied across each slot. The 1:4 serum dilution is applied in the slot marked Ref., and the 1:15 serum dilution in the other 5 slots.
- the sample is allowed to diffuse into the gel for 5 minutes, and then the sample template is blotted gently with a Sample Blotter in order to remove excess sample liquid. The Blotter is discarded, and the Sample Template is carefully removed and discarded.
- the DAKO Electrophoresis Apparatus is filled with 300 mL diluted buffer (150 mL in each compartment) .
- the gel is placed in the apparatus so as to form an arch (gel side down) in such as way that the (-) side of the gel dips into the cathode compartment (-) , and that the (+ ) side of the gel dips into the anode compartment (+ ) .
- the lid is placed on the apparatus and power supply is connected.
- the voltage is set to 120 V and the electrophoresis is continued for 25 minutes.
- the power supply is disconnected, and the gel is carefully removed from the apparatus and placed on a level surface, gel side up.
- the electrophoresis buffer is discarded.
- Immunofixation (specific precipitation of the separated proteins) .
- the surface of the gel is gently blotted with a Gel Blotter.
- the Gel Blotter is removed immediately and discarded.
- the Antibody Template is placed over the surface of the gel so that the troughs of the Template are in alignment with those printed on the plastic backing of the gel. It should be ensured that a close contact between the Template and the surface of the gel is obtained.
- the Template is gently rubbed in order to remove air bubbles.
- Ref. 80 ⁇ L of Protein Fixative Solution
- IgG 80 ⁇ L of Anti-IgG
- IgA 80 ⁇ L of Anti-IgA
- IgM 80 ⁇ L of Anti-IgM
- K 80 ⁇ L of Anti-Kappa
- L 80 ⁇ L of Anti-Lambda. It should be ensured that the volume is evenly distributed within the trough. Furthermore, the surface of the gel should not be touched. The gel is incubated with the Antibody Template in a humid box for 15 minutes at room temperature. Subsequently, the gel is placed on a levelled surface and the Antibody Template is carefully removed.
- Washing and Staining Accessory Kit are filled in the following way: Washing: 1 container with 300 mL Saline Solution. Staining: 1 container with 300 mL diluted Staining Solution. Rinsing: 1 container with 300 L distilled water. Destaining: 1 container with 300 mL acetic acid, 5%.
- the gel is covered with one sheet of Gel Blotter, two sheets of Drying Blotter and a glass plate. The gel is pressed under a weight of approximately 1 kg for 10 minutes. Subsequently, the Blotters are removed and discarded, and the gel is immersed in saline solution and washed for 10 minutes without agitation. The pressing procedure is repeated as previously described. After pressing, the Blotters are discarded, and the gel is dried in a current of hot air or, alternatively, dried in a drying oven (maximum temperature 90°C) for approximately 5 minutes.
- Excess Staining Solution is rinsed off in distilled water before destaining. Destaining is performed in fresh Destaining Solution for approximately 2 minutes, or until the background has a faint blue colour.
- the gel is dried for 5 minutes as previously described or until the gel is completely dry.
- each immunofixation gel shows the results of one patient serum sample.
- the lane at the left is a reference lane showing all serum proteins in the serum sample in the order albumin (top) , followed by alpha, beta, and gamma globulins (immunoglobulins) .
- the following lanes are a visualisation of the patient's immunoglobulins established by the use of specific antibodies. From left to right, the gel shows IgG, IgA, IgM, kappa light chain, and lambda light chain.
- Figure 2 The results shown in Figure 2 are obtained using the sodium benzoate buffer.
- Figure 2A shows a patient having clearly defined double bands in the gamma region. The bands can be identified as IgM, kappa.
- Figure 2B shows a patient having strong double bands in the gamma region. The bands are stronger than in Figure 2A. The bands can be identified as IgM, kappa.
- Figure 2C shows a patient having a strong band which can be identified as IgA, lambda.
- Figure 2D shows a patient having a very strong band which can be identified as IgG, lambda.
- Figure 3 The results shown in Figure 3 are obtained using the ammonium benzoate buffer.
- Figures 3A, 3B, 3C and 3D shows the serum samples as in Figures 2A, 2B, 2C and 2D.
- the buffer used is an ammonium benzoate buffer.
- the results obtained using the ammonium benzoate buffer are not as excellent as the results obtained using the sodium benzoate buffer. However, this might be due to lack of optimisation.
- Example 1 A series of zone electrophoresis and immunofixation procedures using the electrophoresis/immunofixation set- up described in Example 1 were performed.
- the buffer used for this experiment corresponded with regard to the components to the sodium benzoate buffer described in Example 1, however, the gel buffer content of sodium benzoate was varied between 1 and 10 g/L, and the compartment buffer content of sodium benzoate was varied accordingly.
- the separation of the proteins was evaluated and rated 3 (excellent separation) , 2 (acceptable separation) or 1 (bad separation) . The results obtained are shown in the table below.
- Example 1 A series of zone electrophoresis and immunofixation procedures using the electrophoresis/immunofixation setup described in Example 1 were performed.
- the buffer used for this experiment corresponded with regard to the components to the sodium benzoate buffer described in Example 1, however, the gel buffer content of sodium benzoate being 7 g/L, whereas the compartment buffer content of sodium benzoate was varied between 0.8 and 7 g/L.
- the separation of the proteins was evaluated and rated 3 (excellent separation) , 2 (acceptable separation) or 1 (bad separation) . The results obtained are shown in the table below.
Abstract
Description
Claims
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU55225/00A AU5522500A (en) | 1999-06-29 | 2000-06-29 | Benzoate buffers for zone electrophoresis and immunofixation |
EP00940223A EP1192464A1 (en) | 1999-06-29 | 2000-06-29 | Benzoate buffers for zone electrophoresis and immunofixation |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
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DKPA199900935 | 1999-06-29 | ||
DKPA199900935 | 1999-06-29 |
Publications (1)
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WO2001002859A1 true WO2001002859A1 (en) | 2001-01-11 |
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Application Number | Title | Priority Date | Filing Date |
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PCT/DK2000/000350 WO2001002859A1 (en) | 1999-06-29 | 2000-06-29 | Benzoate buffers for zone electrophoresis and immunofixation |
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EP (1) | EP1192464A1 (en) |
AU (1) | AU5522500A (en) |
WO (1) | WO2001002859A1 (en) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
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US7648678B2 (en) | 2002-12-20 | 2010-01-19 | Dako Denmark A/S | Method and system for pretreatment of tissue slides |
CN105699647A (en) * | 2015-09-11 | 2016-06-22 | 中国农业科学院哈尔滨兽医研究所 | Novel electrophoretic buffer solution, and preparation method and application thereof |
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US4321119A (en) * | 1979-11-07 | 1982-03-23 | Gelman Sciences, Inc. | Buffer composition and method for the electrophoretic separation of proteins |
US5891422A (en) * | 1996-10-10 | 1999-04-06 | Warner-Lambert Company | Antimicrobial composition containing a C3 -C6 alcohol |
-
2000
- 2000-06-29 WO PCT/DK2000/000350 patent/WO2001002859A1/en not_active Application Discontinuation
- 2000-06-29 AU AU55225/00A patent/AU5522500A/en not_active Abandoned
- 2000-06-29 EP EP00940223A patent/EP1192464A1/en not_active Withdrawn
Patent Citations (2)
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US4321119A (en) * | 1979-11-07 | 1982-03-23 | Gelman Sciences, Inc. | Buffer composition and method for the electrophoretic separation of proteins |
US5891422A (en) * | 1996-10-10 | 1999-04-06 | Warner-Lambert Company | Antimicrobial composition containing a C3 -C6 alcohol |
Non-Patent Citations (7)
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AHUMADA, I. ET AL: "Determination of organic acids and phosphate in soil aqueous extracts by capillary zone electrophoresis.", COMMUNICATIONS IN SOIL SCIENCE AND PLANT ANALYSIS, vol. 30, no. 1-2, 1999, pages 213 - 220, XP000856533 * |
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CHANKVETADZE, BEZHAN ET AL: "Capillary electrophoresis enantioseparation of noncharged and anionic chiral compounds using anionic cyclodextrin derivatives as chiral selectors", JOURNAL OF CAPILLARY ELECTROPHORESIS, vol. 2, no. 5, 1995, pages 235 - 240, XP000856503 * |
LUNA, E. A. ET AL: "Evaluation of the utility of capillary electrophoresis for the analysis of sulfobutyl ether.beta.-cyclodextrin mixtures", JOURNAL OF PHARMACEUTICAL AND BIOMEDICAL ANALYSIS, vol. 15, no. 1, 1996, pages 63 - 71, XP000856460 * |
See also references of EP1192464A1 * |
TARNAI, M. ET AL: "Capillary electrophoretic separation of mono- and dimaltosyl-.beta.-cyclodextrins and determination of the stability constants of their benzoate complexes.", CHROMATOGRAPHIA, vol. 48, no. 5-6, 1998, pages 383 - 387, XP000856531 * |
TAWALI, A. B. ET AL: "Determination of phytic acid content of soya beans during tempeh production using capillary electrophoresis.", DEUTSCHE LEBENSMITTEL-RUNDSCHAU, vol. 94, no. 1, 1998, pages 28 - 30, XP000856532 * |
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Also Published As
Publication number | Publication date |
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EP1192464A1 (en) | 2002-04-03 |
AU5522500A (en) | 2001-01-22 |
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