US3334969A - Method and apparatus for chromatography analysis and hydrolysis of peptides - Google Patents

Method and apparatus for chromatography analysis and hydrolysis of peptides Download PDF

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Publication number
US3334969A
US3334969A US314812A US31481263A US3334969A US 3334969 A US3334969 A US 3334969A US 314812 A US314812 A US 314812A US 31481263 A US31481263 A US 31481263A US 3334969 A US3334969 A US 3334969A
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United States
Prior art keywords
tubing
peptides
reagent
eluent
heating bath
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Expired - Lifetime
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US314812A
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English (en)
Inventor
Catravas George Nicholas
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Technicon Corp
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Technicon Corp
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Filing date
Publication date
Priority to GB1051309D priority Critical patent/GB1051309A/en
Application filed by Technicon Corp filed Critical Technicon Corp
Priority to US314812A priority patent/US3334969A/en
Priority to DE19641523071 priority patent/DE1523071B2/de
Priority to CH1278664A priority patent/CH469978A/de
Priority to FR990466A priority patent/FR1449605A/fr
Priority to SE12070/64A priority patent/SE316929B/xx
Priority to BE654083D priority patent/BE654083A/xx
Application granted granted Critical
Publication of US3334969A publication Critical patent/US3334969A/en
Anticipated expiration legal-status Critical
Expired - Lifetime legal-status Critical Current

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N31/00—Investigating or analysing non-biological materials by the use of the chemical methods specified in the subgroup; Apparatus specially adapted for such methods
    • G01N31/22—Investigating or analysing non-biological materials by the use of the chemical methods specified in the subgroup; Apparatus specially adapted for such methods using chemical indicators
    • 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
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/74—Optical detectors
    • 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
    • G01N30/84—Preparation of the fraction to be distributed

Definitions

  • the primary object of the present invention is to provide a method and apparatus for the continuous automatic hydrolysis of peptides discharged from a chromatography column and for the automatic quantitative analysis of the hydrolyzed peptides.
  • the apparatus includes a chromatography column 10, to the upper end of which an eluting liquid is supplied from a variable gradient device 12 by means of a positive displacement pump 14 of the cylinder-piston type.
  • the variable gradient device 12 is preferably of the type shown and described in US. Patent No. 3,137,480 issued to lack Isreeli on June 16, 1964.
  • the device comprises a series of chambers, indicated at 34, which are in fluid flow communication with each other at their respective bottoms and which'respectively contain the eluting liquid or buffer of dilferent concentrations.
  • the buffer flows from one chamber to the succeeding chamber and from the device through the bottom outlet 36 from which it is pumped, as before stated, to the top of the column 10 by way of the tubing 38 and 40.
  • the peptides previously adsorbed in the resin of the column prior to the start of the stripping operation, in a manner well understood in the art of chromatography, are discharged in succession through the outlet 42 at the bottom of the column and flow to the pump tubing 44 of the pump 46.
  • the pump 46 also includes pump tube 48 for the supply of sodium hydroxide or other suitable alkali for hdyrolyzing the peptides in the manner presently explained.
  • the pump also includes a pump tube 50 by which nitrogen is introduced into the liquid stream at the tube fitting 52 where the sodium hydroxide is introduced into the fluid stream containing the peptides.
  • the nitrogen segmentizes the liquid so that the fluid stream which enters the tubing 54 consists of segments of liquid separated from each other by segments of nitrogen.
  • Nitrogen is employed for segmentizing the fluid stream instead of alkali-free air because it has been found to increase the percentage of hydrolysis considerably by reason of the fact that it enables a stronger alkali to be used with little or negligible destructive effect on the alkali sensitive amino acids.
  • I employ sodium hydroxide of 16% to 20% by volume in a water solution thereof.
  • the fluid stream flows from the tube 54 through a mixing coil 56 into the tubing 58 of a heating bath 60 in which the peptides are subjected to United States Patent 0 3,334,969 Patented Aug. 8, 1967 Mice the action of the alkali and of the heat in the heating bath, which is maintained at a temperature of C.
  • the tubing of the heating bath is of considerable length, say about feet long measured linearly of the tubing, although it is arranged in a coil-like fashion for space economy in the heating bath receptacle 60 in which the heating liquid is contained.
  • the tubing 58 has an internal diameter of preferably 0.085 inch.
  • Tubing 58 is made of a fluorinated hydrocarbon which in itself is well known and readily available on the market, being designated by the trademark Teflon. Said tubing is resistant to alkalis, even at elevated temperatures, but because of the hydrophobic nature of Teflon, objectionable pressure variations and surging of the fluid in said tubing occur.
  • Said vent consists of a capillary tube formed of polyvinyl chloride. While the length and internal diameter of said tube may vary somewhat, the preferred length is six inches and its internal diameter is 0.005 inch.
  • the vent tube 62 is connected in the alkaline supply line upstream of the fitting 52 in which the alkali and the peptides flow into the tube 54 along with the nitrogen gas whichv segmentizes the fluid stream, as explained above.
  • the outlet of the tubing 58 is connected by tubing 64 to the pump tube 66, and it will be noted that the fluid stream is pumped into the tubing 58 and is also pumped from said tubing.
  • the segmented fluid stream which includes the peptides and the alkali is pumped into the heating bath where hydrolysis of the peptides takes place at a temperature of 95 C., and the hydrolyzed peptides are pumped out of the heating bath concurrently with the flow of the successive peptides from the outlet 42 of the chromatography column to the pump tube 44.
  • Said apparatus includes the pump 46 which may be part of the pump 46 or which may be a separate pump which, like pump 46, is of the compressible tube type.
  • pump 46 or 46 is a proportioning pump preferably of the type described in United States Patent No. 2,935,028 issued May 3, 1960. Briefly described, such pump includes a platen P and a plurality of resiliently compressible tubes which are compressed progressively along their lengths simultaneously by pressure rollers indicated by the circles within the outline of the platen P, one of said rollers being identified by the letter R.
  • the hydrolyzed peptides are transmitted in succession via the pump tube 66, tubes 68 and 70, to pump tube 72.
  • a gas venting tube 74 is interposed in the line of tubing 68, 70, in order to remove the nitrogen segments from the fluid stream before it is supplied to the pump tube 72.
  • Saidv'ent 74 is above the point 67 which connects tube 68 to tube 70 so that the nitrogen rises in vent tube 74 and is vented to atmosphere while the liquid segments floW point 67 into tube 70 and are consolidated into a continuous liquid stream which flows to pump tube 72.
  • the hydrolyzed peptides are analyzed colorimetrically in the analysis apparatus in a known way which will now be briefly described.
  • the peptides are treated for colorimetric analysis by mixing them with a liquid which comprises a diluent such as methyl Cellosolve and a color reagent which is ninhydrin in the presence. of hydrindantin as a reducing agent.
  • This liquid is preferably pre-mixed and is supplied from an opaque container (not shown) to the pump tube 75 which is connected to the inlet of a mixing coil 76.
  • a neutralizing acid, such as acetic acid, is supplied by pump tube 78 to neutralize residual alkali which may not be fully consumed during the hydrolyzing action in the heating bath 60.
  • Nitrogen for segmentizing the fluid stream is supplied by the pump tube 80. It will be noted that the acetic acid is introduced into the hydrolyzed peptides at the fitting 82 upstream of the fitting 84 in order to effect said neutralization before the color reagent is added to the peptides at the inlet of the mixing coil 76.
  • the segmentized fluid stream flows from the mixing coil 76 to the tubing 86 of heating bath 88 which operates at a temperature of 95 C.
  • the outlet of the tubing of the heating bath is connected to a cooling coil 90, the outlet of which is connected by a tube 92 to the colorimeter flow cell 94 at its inlet 96.
  • the liquid outlet of the flow cell is indicated at 98.
  • a tube 100 is connected to the tube 92 upstream of the inlet 96 of the flow cell and to the pump tube 101 so that segments or bubbles of nitrogen introduced into the fluid stream by the pump tube 80, as previously explained, are removed from the fluid stream before it enters the flow cell.
  • the nitrogen bubbles and a small quantity of liquid are thus aspirated from tube 92 so that only liquid enters the flow cell.
  • the light source including its lenses for transmitting the light longitudinally of the flow cell through the liquid during flow therethrough, is indicated at 102.
  • the detector which is responsive to the light leaving the fiow cell after it passes through the liquid, is indicated at 104, and the recorder which is operable under the control of said detector is indicated at 106. It will be understood that the nitrogen introduced by pump tubes 50 and 80 for segmentizing the liquid streams have a cleansing action in that the flow of the nitrogen bubbles through the tubing remove particles which might otherwise adhere to and accumulate on the inner surfaces of the tubing.
  • tube 100 may be connected to the outlet 98 of the flow cell and the gas bubbles allowed to flow directly to waste from the top of fitting 103 when tube 100 is disconnected therefrom.
  • the diameters of the pump tubing upstream of the heating bath 58 and the diameter of tube 66 are such that the fluid in said heating bath is under positive pressure and so that a small quantity of liquid flows continuously out of the vent tube 62.
  • chromatography analysis apparatus comprising a chromatography column, continuously operable means for hydrolyzing the peptides contained in an eluent which is discharged from said column during the stripping operation, said hydrolyzing means comprising a heating bath having tubing through which said peptide-containing eluent and a reagent for hydrolyzing the peptides flow, said tubing being resistant to said hydrolyzing reagent, and pump means and tubing for supplying to said tubing of the heating bath said reagent along with the peptidecontaining eluent as the latter is discharged from the column, said supply tubing comprising separate tubing for said peptide-containing eluent and said reagent, respectively, and a capillary vent tube coupled to the supply tubing for the reagent.
  • chromatography analysis apparatus comprising a chromatography column, continuously operable means for hydrolyzing the peptides contained in an eluent which is discharged from said column during the stripping operation, said hydrolyzing means comprising a heating bath having tubing through which said peptide-containing eluent and a reagent for hydrolyzing the peptides flow, said tubing being resistant to said hydrolyzing reagent, and pump means and tubing for supplying to said tubing of the heating bath said reagent along with the peptidecontaining eluent as the latter is discharged from the column, said supply tubing comprising separate tubing for said peptide-containing eluent and said reagent, respectively, and a capillary vent tube coupled to the supply tubing for the reagent, and means disposed downstream of said capillary vent tube and upstream of said supply tubing to mix said peptide-containing eluent and reagent with each other before they enter the heating bath tubing.
  • chromatography analysis apparatus comprising a chromatography column, continuously operable means for hydrolyzing the peptides contained in an eluent which is discharged from said column during the stripping operation, said hydrolyzing means comprising a heating bath having tubing through which said peptide-containing eluent and a reagent for hydrolyzing the peptides flow, said tubing being resistant to said hydrolyzing reagent, and pump means and tubing for supplying to said tubing of the heating bath said reagent along with the peptidecontaining eluent as the latter is discharged from the column, said supply tubing comprising separate tubing for said peptide-containing eluent and said reagent, respectively, and a capillary vent tube coupled to the supply tubing for the reagent, means operable concurrently with the flow of the liquid from said heating bath for treating the hydrolyzed peptides for colorimetric examination, and means operable concurrently with said treating means for quantitatively analyzing the hydrolyzed peptide
  • chromatography analysis apparatus comprising a chromatography column, continuously operable means for hydrolyzing the peptides contained in an eluent which is discharged from said column during the stripping operation, said hydrolyzing means comprising a heating bath having tubing through which said peptide-containing eluent and a reagent for hydrolyzing the peptides flow, said tubing being resistant to said hydrolyzing reagent, and pump means and tubing for supplying to said tubing of the heating bath said reagent along with the peptidecontaining eluent as the latter is discharged from the column, said supply tubing comprising separate tubing for said peptide-containing eluent and said reagent, respectively, and a capillary vent tube coupled to the supply tubing for the reagent, and means disposed downstream of said capillary vent tube and upstream of said supply tubing to mix said peptide containing eluent and reagent with each other before they enter the heating bath tubing, means operable concurrently with
  • Hydrolysis apparatus comprising a heating bath having a length of tubing for the flow therethrough and the heating of a stream of fluid for the hydrolysis process, pump means and tubing for supplying to said tubing of the heating bath a stream of fluid containing a substance to be hydrolyzed and a reagent to hydrolyze said substance, said tubing of the heating bath being resistant to said hydrolyzing reagent, said supply tubing comprising separate tubing for said substance and said reagent, rc-
  • a capillary vent tube coupled to the supply tubing for the reagent.
  • Hydrolysis apparatus comprising a heating bath having a length of tubing for the flow therethrough and the heating of a stream of fluid for the hydrolysis process, pump means and tubing for supplying to said tubing of the heating bath a stream of fluid containing a substance to be hydrolyzed and a reagent to hydrolyze said substance, said tubing of the heating bath being resistant to said hydrolyzing reagent, said supply tubing comprising Separate tubing for said substance and said reagent, respectively, and a capillary vent tube coupled to the supply tubing for the reagent, and means disposed downstream of said capillary vent tube and upstream of said substance-supply tubing to mix said substance and reagent with each other before they enter the heating bath tubing.
  • stripping operation comprising a heating bath having a length of tubing for the flow therethrough and the heating of a stream of eluent for the hydrolysis process, pump means and tubing for supplying to said tubing of the heating bath a stream of said eluent and a reagent to hydrolyze said eluent, said tubing of the heating bath being resistant to said hydrolyzing reagent, said supply tubing comprising separate tubing for said eluent and said reagent, respectively, and a capillary vent tube coupled to the supply tubing for the reagent.
  • means for hydrolyzing hydrolyzable substances present in eluent being discharged from a chromatography column during the stripping operation comprising a heating bath having a length of tubing for the flow therethrough and the heating of a stream of eluent for the hydrolysis process, pump means and tubing for supplying to said tubing of the heating bath a stream of said eluent and a reagent to hydrolyze said eluent, said tubing of the heating bath being resistant to said hydrolyzing reagent, said supply tubing comprising separate tubing for said eluent and said reagent, respectively, and a capillary vent tube coupled to the supply tubing for the reagent, and means disposed downstream of said capillary vent tube and upstream of said heating bath to mix said eluent and said reagent with each other before they enter said tubing of the heating bath.
  • the method which comprises stripping the column containing the peptides by transmitting an eluent downwardly through the column and discharging the peptide containing eluent from the bottom of the column, concurrently forming in a tubing a liquid stream in which said dis charged eluent and peptide alkaline-hydrolyzing reagent are present, introducing nitrogen into said stream thereby forming a fluid stream in said tubing comprising spaced segments of said liquid separated from each other by intervening segments of nitrogen, and passing said segmented stream through a heating bath to hydrolyze said peptides by the action of heat and said reagent thereon.
  • the method which comprises stripping the column containing the peptides by transmitting an eluent downwardly through the column and discharging the peptide containing eluent from the bottom of the column, concurrently forming in a tubing a liquid stream in which said discharged eluent and peptide alkaline-hydrolyzing reagent are present, introducing nitrogen into said stream thereby forming a fluid stream in said tubing comprising spaced segments of liquid separated from each other by intervening segments of nitrogen, and passing said segmented stream through a heating bath to hydrolyze said peptides by the action of heat and said reagent thereon, transmitting the stream from said heating bath through tubing from which said nitrogen segments are removed thereby forming a continuous liquid stream containing said hydrolyzed peptides, concurrently treating said last mentioned stream for colorimetric analysis thereof in respect to said peptides in successive portions of said last mentioned stream, and concurrently examining the liquid of said treated stream
  • the method which comprises stripping the column containing the peptides by transmitting an eluent downwardly through the column and discharging the peptide containing eluent from the bottom of the column, concurrently forming in a tubing a liquid stream in which said discharged eluent and peptide alkaline-hydrolyzing reagent are present, introducing nitrogen into said stream thereby forming a fluid stream in said tubing comprising spaced segments of said liquid separated from each other by intervening segments of nitrogen, and passing said segmented stream through a heating bath to hydrolyze said peptides by the action of heat and said reagent thereon, said reagent being supplied through a separate tube to said tubing by pump means which transmits said discharged eluent to said tubing, and passing said liquid stream through alkaline resistant tubing of a heating bath while providing a reduction in the rate of supply of said reagent in response to an increase in the backpressure at said heating bath

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  • Life Sciences & Earth Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Spectroscopy & Molecular Physics (AREA)
  • Peptides Or Proteins (AREA)
  • Sampling And Sample Adjustment (AREA)
  • Automatic Analysis And Handling Materials Therefor (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
US314812A 1963-10-08 1963-10-08 Method and apparatus for chromatography analysis and hydrolysis of peptides Expired - Lifetime US3334969A (en)

Priority Applications (7)

Application Number Priority Date Filing Date Title
GB1051309D GB1051309A (xx) 1963-10-08
US314812A US3334969A (en) 1963-10-08 1963-10-08 Method and apparatus for chromatography analysis and hydrolysis of peptides
DE19641523071 DE1523071B2 (de) 1963-10-08 1964-09-30 Verfahren und vorrichtung zur chromatographischen analyse von peptiden
CH1278664A CH469978A (de) 1963-10-08 1964-10-01 Verfahren und Vorrichtung zur Analyse von Peptiden
FR990466A FR1449605A (fr) 1963-10-08 1964-10-06 Procédé et appareil pour analyse chromatographique et hydrolyse de peptines
SE12070/64A SE316929B (xx) 1963-10-08 1964-10-07
BE654083D BE654083A (xx) 1963-10-08 1964-10-07

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Application Number Priority Date Filing Date Title
US314812A US3334969A (en) 1963-10-08 1963-10-08 Method and apparatus for chromatography analysis and hydrolysis of peptides

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US3334969A true US3334969A (en) 1967-08-08

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US (1) US3334969A (xx)
BE (1) BE654083A (xx)
CH (1) CH469978A (xx)
DE (1) DE1523071B2 (xx)
GB (1) GB1051309A (xx)
SE (1) SE316929B (xx)

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3458285A (en) * 1965-03-26 1969-07-29 Ceskoslovenska Akademie Ved Method of reducing the undesirable decrease of the concentration gradients
US3508880A (en) * 1966-01-07 1970-04-28 Ceskoslovenska Akademie Ved Apparatus for carrying out chromatographic analyses of amino acids,their mixtures and similar materials
US3515491A (en) * 1966-10-27 1970-06-02 Gilford Instr Labor Inc Fluid sample flow cell
US3531258A (en) * 1967-11-16 1970-09-29 Us Health Education & Welfare Apparatus for the automated synthesis of peptides
US3649203A (en) * 1968-11-22 1972-03-14 Ralston Purina Co Automatic analyzer
US3804593A (en) * 1964-05-25 1974-04-16 Technicon Instr Automatic analysis apparatus and method
US3892532A (en) * 1973-10-24 1975-07-01 Hoffmann La Roche Fluorescamine peptide analyzer
US3929413A (en) * 1974-03-04 1975-12-30 Anatronics Corp Fluid transport and metering system
US4049381A (en) * 1976-03-23 1977-09-20 Technicon Instruments Corporation Apparatus and method of fluid sample analysis
US4540548A (en) * 1977-04-07 1985-09-10 Yamanouchi Pharmaceutical Co., Ltd. Method of detecting fluorescent materials and apparatus for their detection
US4755558A (en) * 1986-05-30 1988-07-05 Beckman Instruments, Inc. Using internal marker
US4855486A (en) * 1986-05-30 1989-08-08 Kalbag Suresh M Blocked, marked amino acids

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4470699A (en) * 1982-08-12 1984-09-11 The United States Of America As Represented By The United States Department Of Energy Micro-column plasma emission liquid chromatograph

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3116754A (en) * 1959-03-17 1964-01-07 Technicon Instr Fluid treatment apparatus

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3116754A (en) * 1959-03-17 1964-01-07 Technicon Instr Fluid treatment apparatus

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3804593A (en) * 1964-05-25 1974-04-16 Technicon Instr Automatic analysis apparatus and method
US3458285A (en) * 1965-03-26 1969-07-29 Ceskoslovenska Akademie Ved Method of reducing the undesirable decrease of the concentration gradients
US3508880A (en) * 1966-01-07 1970-04-28 Ceskoslovenska Akademie Ved Apparatus for carrying out chromatographic analyses of amino acids,their mixtures and similar materials
US3515491A (en) * 1966-10-27 1970-06-02 Gilford Instr Labor Inc Fluid sample flow cell
US3531258A (en) * 1967-11-16 1970-09-29 Us Health Education & Welfare Apparatus for the automated synthesis of peptides
US3649203A (en) * 1968-11-22 1972-03-14 Ralston Purina Co Automatic analyzer
US3892532A (en) * 1973-10-24 1975-07-01 Hoffmann La Roche Fluorescamine peptide analyzer
US3929413A (en) * 1974-03-04 1975-12-30 Anatronics Corp Fluid transport and metering system
US4049381A (en) * 1976-03-23 1977-09-20 Technicon Instruments Corporation Apparatus and method of fluid sample analysis
US4540548A (en) * 1977-04-07 1985-09-10 Yamanouchi Pharmaceutical Co., Ltd. Method of detecting fluorescent materials and apparatus for their detection
US4755558A (en) * 1986-05-30 1988-07-05 Beckman Instruments, Inc. Using internal marker
US4855486A (en) * 1986-05-30 1989-08-08 Kalbag Suresh M Blocked, marked amino acids

Also Published As

Publication number Publication date
CH469978A (de) 1969-03-15
BE654083A (xx) 1965-04-07
SE316929B (xx) 1969-11-03
DE1523071B2 (de) 1971-07-29
DE1523071A1 (de) 1969-04-03
GB1051309A (xx)

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