WO1990010873A1 - Puffer- und reagenzsystem für die trennung von aminosäuren - Google Patents

Puffer- und reagenzsystem für die trennung von aminosäuren Download PDF

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Publication number
WO1990010873A1
WO1990010873A1 PCT/EP1990/000384 EP9000384W WO9010873A1 WO 1990010873 A1 WO1990010873 A1 WO 1990010873A1 EP 9000384 W EP9000384 W EP 9000384W WO 9010873 A1 WO9010873 A1 WO 9010873A1
Authority
WO
WIPO (PCT)
Prior art keywords
buffer
reagent
amino acids
ninhydrin
buffers
Prior art date
Application number
PCT/EP1990/000384
Other languages
German (de)
English (en)
French (fr)
Inventor
Hans-Peter Kabus
Original Assignee
MERCK Patent Gesellschaft mit beschränkter Haftung
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by MERCK Patent Gesellschaft mit beschränkter Haftung filed Critical MERCK Patent Gesellschaft mit beschränkter Haftung
Priority to JP2504200A priority Critical patent/JPH04505754A/ja
Publication of WO1990010873A1 publication Critical patent/WO1990010873A1/de

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Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/36—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving ionic interaction, e.g. ion-exchange, ion-pair, ion-suppression or ion-exclusion
    • B01D15/361—Ion-exchange
    • B01D15/362—Cation-exchange
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/42—Selective adsorption, e.g. chromatography characterised by the development mode, e.g. by displacement or by elution
    • B01D15/422—Displacement mode
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J39/00—Cation exchange; Use of material as cation exchangers; Treatment of material for improving the cation exchange properties
    • B01J39/26—Cation exchangers for chromatographic processes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography

Definitions

  • the invention relates to a buffer and reagent system for the elution and separation of primary and secondary amino acids and other ninhydrin-positive compounds on the cation exchanger with lithium, sodium, or potassium, phosphate.
  • Buffer solutions containing acetate, borate and / or formations, which differ in pH and molarity, and on a ninhydrin-containing reagent for the post-column reaction It is suitable for the separation of hydrolyzate and physiological amino acids, for various amines and polyamines and their qualitative and quantitative determination.
  • a sodium buffer system is used to separate hydrolyzate amino acids. Physiological amino acids can only be successfully separated with a lithium buffer system.
  • FIG. 1 A functional diagram is shown in Figure 1.
  • FIG. 2 A functional diagram is shown in Figure 1.
  • FIG. 1 The structure of today's amino acid analyzer is shown in Figure 2.
  • the reagent ninhydrin is still dissolved in the toxic methyl cellosolve (ethylene glycol monomethyl ether) (United States Patent 3,537,821), which is mixed with a high molarity sodium or lithium acetate buffer in a ratio of 2: 1 to 3: 1.
  • the reagent buffer with pH 5.5 and its high molarity should buffer the acidic or alkaline pH of the solutions required for the separation to pH 5.5.
  • the reagent buffer ensures the optimal pH of the reaction with ninhydrin.
  • a reducing compound is added to the reagent, formerly tin-II-chloride, today mostly titanium-III-chloride, which sets the ratio of reduced to oxidized ninhydrin necessary for the reaction with primary and secondary amino groups.
  • This buffer and reagent system is still used in our modern amino acid analyzers in a hardly modified form.
  • the reagent which is quite toxic and unstable in its previous form, tends to precipitate during the derivatization reaction, which as oxide sludge or poorly soluble products block the reactor and can contaminate the assemblies following the reactor. Sensitive quantitative measurements are thereby restricted or made impossible.
  • the object of the invention is to provide a non-aggressive buffer system which does not attack steel or heavy metals, and a stable, less toxic post-column reagent which is tailored to the buffer system and does not derivatize without disturbing residues, the system, in the coordinated combination, not the parts of the analyzer damage or impair its function.
  • the problem is essentially solved in that the buffer solutions are free of citrate and chloride and the reagent in potassium. sodium or lithium acetate, phenolic or pure ethylene glycol is dissolved.
  • the anions of the buffers do not attack steel parts and considerably increase the effectiveness and service life of the steel separation columns and other steel or metal parts of the amino acid analyzer.
  • the reagent according to the invention contains less toxic ethylene glycol and a potassium acetate buffer, which shows better solubility for organic compounds. Phenol as an organic additive increases the stability of the reagent and additionally the solubility of the products formed in the derivatization. The problem mentioned at the outset of possible blockage of the reactor and contamination of the photometer cuvette, as well as its subsequent parts, is thus solved.
  • buffers proposed according to the invention and the reagent can also be used separately.
  • Table 1 shows the composition of the buffers and the reagent.
  • Buffers A to C are suitable for the separation of hydrolyzate amino acids. Buffers that separate physiological amino acids on the cation exchanger are set differently in terms of molarity and pH. The advantages and the effects of the invention are explained below.
  • the buffers according to the invention are composed of formate, acetate and phosphate buffers. They have all the properties of the usual citrate buffers. They can be set in the pH range from pH 2.2 to 12. In order to obtain optimal buffering, formate buffers are used in the range 2.2 to 3.5, in the range between 3.5 and 5.5 pH acetate buffers and in the range from 5.5 to 12 pH phosphate buffers.
  • the pH of these buffers is adjusted with formic acid, acetic acid, sulfuric acid, phosphoric acid or, as required, from a mixture of the acids mentioned. No chloride or citrate anions are used.
  • the buffers are protected against microbial growth with sodium azide or octanoic acid.
  • borate acetate buffers must be used in the pH range between 5.5 and 12, since lithium phosphate is sparingly soluble.
  • the pH values of the buffers to be set depend on the separation task and the separation resins used.
  • All buffers according to the invention used for amino acid analysis contain no citrate and no halide ions. They are mild buffers that do not attack the stainless steel parts of the amino acid analyzer and only dissolve heavy metals in insignificant amounts.
  • buffers and the reagent can also be used in combination for high-pressure liquid chromatography components (HPLC), which are predominantly made up of stainless steel parts and are connected to stainless steel capillaries. It is even possible to use a wound stainless steel capillary for the post-column reactor, which guarantees a significantly better heat transfer compared to plastic capillaries.
  • the capillary length can also be shortened due to the rapid heat exchange. This leads to less band broadening of the amino acid peaks eluted from the cation exchange column. The peaks become sharper and higher. This increases detection sensitivity, for example for amino acids. Such a reactor has been under test for twelve months.
  • buffer jumps den increase in the baseline
  • Buffer jumps make it more difficult for the computer to evaluate an amino acid chromatogram.
  • the buffers mixed with formic acid have a different refractive index than the buffers mixed with acetic acid and most UV-VIS detectors react to different refractive indices of the media flowing through the cuvette. More free formic acid in the buffer increases the baseline, more free acetic acid lowers the baseline. This compensation option is not available when using the citrate buffers previously used.
  • the ninhydrin reagent according to the invention is composed of 40-50% potassium acetate buffer, 50-40% ethylene glycol and 10% phenol.
  • the 4N potassium acetate buffer with pH 5.5 has less tendency to crystallize out in the cold than the sodium acetate buffer previously used. It has better solution properties for those formed during the ninhydrin reaction organic substances (the potassium ion has a weaker and more easily displaceable hydration shell than the lithium or sodium ion).
  • the above-mentioned mixing ratio between potassium acetate buffer and ethylene glycol leads to a stronger buffering with the addition of regeneration lye.
  • the reagent pH changes little. As a result, no red-brown or similar precipitates form that could clog the coil.
  • the non-toxic ethylene glycol used for the reagent contains fewer or no peroxides that limit the shelf life of the reagent.
  • the ninhydrin reagent can remain in the reactor over a longer period of time without any disturbances occurring: an essential advantage over commercially available ninhydrin reagents.
  • the long-term tests were carried out over several days using the LC 5001 and LC 3000 amino acid analyzer.
  • the phenol addition causes a better solubility for aromatic compounds e.g. for the blue reaction product formed with ninhydrin and amino acids.
  • Overloading the separation column with nin ⁇ hydrine-positive compounds and their subsequent strong reaction with reagent no longer lead to precipitation of the reaction products in the coil.
  • the reactor and the cuvette of the photometer remain clean.
  • Hydrindantine as a reducing agent does not lead to poorly soluble oxides when the reagent is oxidized by oxygen or peroxides, as when tin (II) chloride or titanium III chloride is added. No oxide sludge is formed. Reagent lines, reactor coil and photometer cuvette remain clean. Small amounts of hydrazine sulfate stabilize the reagent and increase the shelf life. The oxygen-free reagent can be stored for months in sealed brown glass bottles.
  • the invention relates to a citrate- and chloride-free buffer and reagent system for the separation of amino acids and other ninhydrin-positive components on the cation exchanger.
  • the buffers contain formate, acetate or phosphate anions and their free acids. They do not contain chloride or citrate anions.
  • the reagent consists of a potassium, sodium or lithium acetate buffer, ninhydrin, hydrindantine and phenol. Ethylene glycol is used as the solvent.
  • the buffer and reagent system according to the invention increases the service life of amino acid analyzers in that stainless steel parts are not corroded, separating resins packed in steel columns are not poisoned and overloading the reactor with amino acid derivatives is tolerated much better. Valves and reactors are not blocked by oxide sludge and the photometer cuvette and the subsequent components are not contaminated by precipitation. Because of these properties, the buffer and reagent system is particularly suitable for conventional HPLC systems. Buffer and reagent specification for the hydrolyzate program
  • Reagent composition 20 g ninhydrin

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  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Organic Chemistry (AREA)
  • Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Investigating Or Analyzing Non-Biological Materials By The Use Of Chemical Means (AREA)
PCT/EP1990/000384 1989-03-11 1990-03-09 Puffer- und reagenzsystem für die trennung von aminosäuren WO1990010873A1 (de)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2504200A JPH04505754A (ja) 1990-03-09 1990-03-09 アミノ酸類を分離するための緩衝液及び反応剤の系

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DEP3907930.9 1989-03-11
DE19893907930 DE3907930C2 (de) 1989-03-11 1989-03-11 Puffer- und Reagenzsystem für die Trennung von Aminosäuren

Publications (1)

Publication Number Publication Date
WO1990010873A1 true WO1990010873A1 (de) 1990-09-20

Family

ID=6376104

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP1990/000384 WO1990010873A1 (de) 1989-03-11 1990-03-09 Puffer- und reagenzsystem für die trennung von aminosäuren

Country Status (3)

Country Link
EP (1) EP0427807A1 (enrdf_load_stackoverflow)
DE (1) DE3907930C2 (enrdf_load_stackoverflow)
WO (1) WO1990010873A1 (enrdf_load_stackoverflow)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3686118A (en) * 1971-01-11 1972-08-22 Durrum Chem Corp Chromatographic method
US3778230A (en) * 1970-12-25 1973-12-11 H Sagusa Method of automatically analyzing amino acids by liquid chromatography and color-developing solution therefor
US4274833A (en) * 1979-10-18 1981-06-23 Pickering Michael V Ninhydrin reagent for use in amine and amino acid analyses

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE1598205B1 (de) * 1964-04-13 1971-05-19 Ceskoslovenska Akademie Ved Einrichtung zur chromatographie von aminosaeuren und derglei chen enthaltenden gemischen
US3537821A (en) * 1967-04-25 1970-11-03 Ceskoslovenska Akademie Ved Method of examining mixtures of amino acids by chromatography
US3649203A (en) * 1968-11-22 1972-03-14 Ralston Purina Co Automatic analyzer
US3897309A (en) * 1974-02-15 1975-07-29 Merck & Co Inc Process for removing pyrogenic material from aqueous solutions
US4274834A (en) * 1979-04-11 1981-06-23 Foote Mineral Company Process for purification of lithium chloride

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3778230A (en) * 1970-12-25 1973-12-11 H Sagusa Method of automatically analyzing amino acids by liquid chromatography and color-developing solution therefor
US3686118A (en) * 1971-01-11 1972-08-22 Durrum Chem Corp Chromatographic method
US4274833A (en) * 1979-10-18 1981-06-23 Pickering Michael V Ninhydrin reagent for use in amine and amino acid analyses

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CHEMICAL ABSTRACTS, Vol. 96, No. 26, 28 June 1982, (Columbus, Ohio, US), D.M. LeMASTER et al., "Prediction of the pH Dependence of the Separation by Ion-exchange : Amino Acid Chromatography With Competitive Buffer Ion Eluents", Abstract 223857t; & ANAL. BIOCHEM., 1982, 122(2), 223-37. *
DIALOG INFORMATIONAL SERVICE, File 351, WPI, Dialog Accession No. 4406058, WPI Accession No. 87-062697/09, G. DROCHIOIU, "Protein Identification and Quantitive Determn. by Hydrolysis With Hydrochloric Acid and Amino-acid Treatment With Diluent Contg. Ninhydrin, Cadmium Nitrate, Acetate Buffer and Glycol"; & RO,A,90 243 (STAT AGRICOLE SUCEA), 30 September 1986. *

Also Published As

Publication number Publication date
DE3907930C1 (enrdf_load_stackoverflow) 1990-06-13
DE3907930C2 (de) 1994-02-24
EP0427807A1 (de) 1991-05-22

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