DE2148603A1 - Fluorimetric determination of primary amino groups - Google Patents

Description

Dr. ! ng, A. τση der Werft - "·· *" * ··· '"

Dr. Fran / Luderef "'" "'" -

2 & Sep. 197!

2U8603

RAH 4090/27

F. Hoffinann-La Roche & Co. Aktiengesellschaft, Bascl / Schwcfe

Fluorometric determination of primary amino groups

The use of ninhydrin as a reagent in colorimetry for the detection and determination of amino acids, amines and peptides has been known for nearly 60 years. Recently, the ninhydrin color reaction has found wide application in context with the newly developed amino acid analyzers for the automatic microanalysis of the amino acid composition of Protein hydrolyzates found. Through the use of micro. techniques it has become possible to use these colorimetric processes using ninhydrin up to the concentration range of nanomoles. This concentration range however, represents the limit of sensitivity for this colorimetric ^ Technology.

It is also known that ninhydrin with compounds containing amino groups gives highly fluorescent products. For example, it is based on the work of McCaman and

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Robins in J. Lab. Clin. Med. ^ G, 885 (1962) pointed out.

It has now been found that arylalkylaldehydes are very strong form fluorescent complexes with ninhydrin, and the present The invention relates to a method for the determination of primary amino groups. Compounds in a mixture, thereby characterized in that this mixture with ninhydrin and an arylalkylaldehyde of the formula

) _n CHO

where Ar is an aromatic radical and η is an integer from 1-8,

heated and the resulting fluorescent substance fluorometrically certainly.

Particularly preferred are aldehydes of the above formula in which η is an integer from 1-3. Most preferred Arylalkylaldehydes are those in which η is the number 1 and the aromatic radical is phenyl or alkyl-substituted phenyl, such as for example phenyl acetaldehyde or 4-methylphenyl acetaldehyde. A very particularly preferred aldehyde is phenylacetaldehyde.

For example, the fluorescence produced under comparable reaction conditions is 3 to 30 ^flia l more pronounced, depending on the pH, if phenylacetaldehyde is used instead of n-butyraldehyde. A large number of compounds containing primary amino groups can be determined by the present method. These types of compounds include, for example, primary amines such as ethylamine, ethanolamine, tryptamine, dopamine, norepinephrine, etc .; Ot-amino acids such as glycine, alanine, leucine, valine, etc. and example

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wise peptides that contain a primary amino group, such as Insulin, angiotensin, leucylalanine, etc.

Of particular importance is that ammonia, secondary and tertiary amines and compounds that contain an amino group as part of a larger functional group, such as amides, Containing guanidines or urethanes, only provide complexes which are extremely low or none at all Have fluorescence and therefore do not interfere with the determination of compounds containing primary amino groups.

The ratio in which the reagents are used is not particularly critical. Usually however preferable when the aldehyde and the ninhydrin in relation to the substance which contains and determines a primary amino group is to be used in large excess, so that the fluorescent product is certainly quantitative Way is formed. In general, an excess of ninhydrin compared to the arylalkylaldehyde is also preferable. At the it is best to use a tenfold excess.

The incubation temperature is also not critical Parameters and can be varied over a temperature range from approximately 40 ° C. to the boiling point of the reaction mixture. However, a temperature range of approximately 55 ° C. is preferred used up to about 65 C. The incubation time varies in Depending on the substrate and the pH, it is usually in a range between 5 minutes and approximately 4 hours. The pH of the incubation medium is usually between about 4 and 9. A pH range of about 6-8 is particular preferred.

The pH is controlled by adding a buffer to the reaction mixture. Suitable buffers are phosphate buffers and other commonly used buffers.

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The selection of a specific pH value for a specific determination depends on numerous factors, such as the substrate and the determination method used. For example, at pH values around 6.0, a stronger fluorescence is observed for amines and peptides than for neutral ones Amino acids. Although in general the absolute intensity of the fluorescence is somewhat lower at lower pH, however, this area offers considerable specificity for the determination of amines and peptides. If the pH value is increased, for example from pH 6 to pH 8, the fluorescence α value of amino acids becomes relative in comparison to amines and peptides elevated. In addition, the incubation time can be shortened if the pH value is increased.

The detection limit of the determination method according to the present invention can be determined for numerous substrates and compared with the detection limit for the standard colorimetric ninhydrin determination method according to Moore and Stein, J. Biöl. Chem. 176 , 367 (1948). The smallest amount that can be detected by the present method is in the range of 10 ~ to · 10 ~ moles, while the colorimetric

-Q see method only allows the detection of 10 ■ * moles to be determined as the smallest amount. Accordingly, the fluorescence-ninhydrin-aldehyde determination method according to the present invention is at least 10 to 100 times more sensitive than the ninhydrin color reaction.

In general, the fluorometric detection is more advantageous compared to the colorimetric determination, since the fluorometric The determination method delivers absolute measured values, while the colorimetric method only provides the difference two measured values are registered, thus reducing the accuracy and the minimum concentration to be determined. Accordingly the limiting factor in a fluorescence determination is only the sensitivity of the detection system, i.e.

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of the instrument used.

2U8603

The determination method according to the present invention was used for the qualitative and quantitative determination of compounds containing primary amino groups in a sample stream automated. It proves to be particularly useful when the continuously flowing eluate from a sample stream Chromatographic system is used, for example a chromatography column for the separation of amino acids.

In such a method, the sample stream is fed into an automatic analyzer at a constant speed j initiated and the reagent stream (ninhydrin, arylalkylaldehyde and buffer) is added. A suitable device A dosing pump is used to control the supply of reagents and the flow rate of the sample stream. after the Reagents are supplied to the sample stream, they are carefully mixed with the sample stream, for example by guides them through a mixing spiral. The mixture is then heated to a suitable temperature for a predetermined time, so that the fluorescent substance can form. The heating is expediently carried out by passing the mixture through a spiral of a certain length, which is immersed in a temperature-constant heating bath, so that the dwell time in the heated spa is J real can be calculated. After the solution has left the heated coil, it is cooled and placed in a flow cell a fluorescence photometer, in which the fluorescence is excited and continuously measured. If desired the detector of the fluorescence photometer can be combined with a suitable recorder, display device or printer. .

Another possible application for the determination method according to the present invention is the qualitative and. quantitative determination of primary amino groups containing

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bindings in a mixture in which one can use a large number of test samples determined successively in a likewise automated manner. · '

In such methods, for example, a probe is immersed into the sample solution and withdraws a certain volume from it. The sample stream withdrawn is as described above processed further in the automatic analyzer. The individual analysis samples taken with the probe are stored within the Sample stream separated from one another in the usual and known manner, by using, for example, solvents or gas bubbles. In the event that the clear width of the pipe system, through which the sample stream flows is sufficiently small, no additional measures are required to separate the individual Sample solutions from each other are required, since the capillary forces are already sufficient to effectively prevent mixing.

The introduction of the sample stream into the analyzer is accomplished using well known techniques.

The automatic determination method according to the present The invention can also be applied to circuit-like control systems for monitoring the content of primary amino groups Compounds are applied in a reaction mixture and so that the course of the reaction can be controlled by methods, as described, for example, by Hana et al. in U.S. Patent 3,306,096.

Another important area of application for the present The invention consists in the determination of compounds containing primary amino groups in the field of paper or Thin layer chromatography. In such a method, the sample is applied to the paper or the thin-layer plate and develop the chromatogram in the usual manner. That Paper or the thin-layer plate is then treated with a solution

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which contains ninhydrin, the arylalkylaldehyde and the pH buffer, as described above for the determinations in solution. The paper or the thin-layer plate is then heated and the fluorescent spots are measured. Instead of the with provisions Any excess of ninhydrin over the arylalkylaldehyde that is used in solution is preferably used the paper or thin-layer chromatography method of determination, an excess of arylalkylaldehyde over the ninhydrin is expedient used. An arylalkylaldehyde to ninhydrin molar ratio of about 3 to 1 is particularly preferred. on in this way, the fluorescent product is formed in high yields. The choice of solvent, pH, the The temperature and the time are dictated by the same considerations as indicated above for the determinations in solution. were asked. Apart from the detection reaction, the usual and are used in paper and thin-layer chromatography known techniques for use (e.g. plotting the sample, developing the chromatogram, etc.).

The following can be cited as examples of the particular advantages of the present invention:

The sensitivity of the inventive determination j method is at least 10 to 100 times higher than the commonly used colorimetric methods and 3 to 30 times higher than with the fluorometric methods for the determination of amines.

The method according to the invention has a wide range of applications and has been applied to a wide variety of primary amines, amino acids and peptides.

The present method is ideally suited for automation, since the fluorescence over a very large Konaentraticnsbereich the concentration of amine substrate proportionally

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nal is.

The analytical technique according to the invention achieves maxi- ■ male sensitivity, since a Beagens is assumed, ·,. ;; ■ that does not fluoresce and the fluorescence only after interaction with a compound 'which has a primary amino group stops, enters. The fact that ammonia the invention Determination method does not interfere in any way, is of the utmost importance, as it is known that protein hydrolyses are always essential Amounts of ammonia contain and also traces of ammonia ■ are constantly present in the atmosphere, making the known

colorimetric and fluorometric methods for amine determination in their scope and in their 'accuracy always' are impaired.

Another advantage of the present method can be seen in the fact that at low pH values, particularly at pH values of around 6, fluorescence occurs with peptides that are higher than with amino acids. So you can see peptides, especially large peptides, which by means of the classic ninhydrin method only provide little colorimetrable color, can be determined with the present method in an excellent manner. A comparison of proteins by means of the so-called "fingerprint method ^fl usually requires several milligrams of protein to determine all peptides. By means of ^". The determination method according to the invention gives the same result when using only microgram amounts of protein. The automated fluorometric determination of peptides, such as those obtained by digesting microgram amounts of protein, enables genetic tests to be carried out on even as small amounts of irotein as they are can be obtained from a single test animal.

Another important application of the present invention is the determination of small amounts physiologically active peptides in cell tissues. In addition to determination and

Identifying known peptides is useful for the present Invention also for the detection of new peptides, their biological Role or activity could not yet be assigned.

In the examples below, all temperatures are given in C.

- ■ .Os;

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example 1 General method for fluorometric determination in solution

2.0 ml of a 0.2 molar phosphate buffer (pH 6, 7 or 8), 0.2 ml of aqueous ninhydrin (50 mmol), 0.1 ml of an aqueous sample solution ( generally

-4 -2
mean 10 to 10 mmol) and finally 0.1 ml of a solution of phenylacetaldehyde in ethanol (10 mmol). After careful mixing, the test tube is covered with an aluminum foil and placed in a 60 ° warm water bath for a suitable incubation time (for example 15 minutes at pH 8, 60 minutes at pH 7 or 120 minutes at pH 6). The test tubes are then cooled in an ice bath for a few minutes, left at room temperature for 10 minutes, and then the fluorescence is measured within one hour (using an Aminco-Beckman fluorophotometer with a 1 cm cell). A wavelength of 390 ηιμ was used for excitation and the emission was measured at 490 πιμ.

Example 2

This example illustrates the relative fluorescence at different ninhydrin concentrations:

High concentration of ninhydrin

Ninhydrin
2250 Phe-CH ₂ CHO
200 Relative
fluorescence Leu-ala
50 + + 820 + - 1 ; + + + 2 -

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2148803

Le u- Ala Ninhydrin Phe-CHpCJf © \
50 100. 100 ■ \ Relative. \
Fluorescence ί Φ Φ Φ \ 10 i
1 \

The fluorescence is given in arbitrary units "The reagent mentioned below each numerical value corresponding to the respective ibj (MoI that in Figure 2, are included k ml of the reaction mixture. The general method, as described in Example 1 was used to ' Develop and measure fluorescence.

example J>

This example illustrates the influence of some aldehydes on the fluorescence of a peptide and ninhydrin:

Aldehydes Relative fluorescence at pH formaldehyde 6.0 7.0 SyO acetaldehyde 11 0 0 . n-butyraldehyde ^: 3 7th 7 y ι ■ . ..Isobutyraldehyde 34 521 349 j _ Ϊ Glutaraldehyde 11 1 0 Benzaldehyde 22nd 2Ϊ 0 trans -cinnamaldehyde i 14th 0 0 ■ ') V Phenylacetaldehyde j 13th 1 Ό Dipheny / acetaldehyde 982 I6II l6O9: 4-methylhydrotropaaldehyde - 0 '; - . 0

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Relative fluorescence at pH Aldehydes 6.0 7.0 8th , 0 4-methylphenylacetaldehyde - 986 - 3,4-dimethoxyphenylacetaldehyde - 0 -

20 μ 1 of each aldehyde in ethanol solution (10 niMol) become a mixture containing 40 μΐ of leucylalanine glycylvaline (1.25 "mmol), 75 μΐ ninhydrin (30 mmol-1) and 200μΐ a 0.6 mo- *" contains laren phosphate buffer at the specified pH. After an incubation time of 90 minutes at 60, the Samples are cooled to room temperature and the fluorescence is measured as described in Example 1.

Example 4

This example illustrates the difference in fluorescence as seen with glycine and a number of glycine peptides is obtained:

Fluorescence 7.0 PH link 6.0 618 8.0 Glycine 81 1159 800 Di-glycine '■ 514 1048 796 Tri-glycine 509 997 710 Tetra-olycin 504 970 620 Penta-glycine 500 580

A mixture of 20) i 1 ethanolic phenylacetaldehyde solution (10 mmol), 37.5) {1 of an aqueous solution of each peptide (13.3 mmol), 75) of aqueous ninhydrins (30 mmol) and 187.5μΐ phosphate buffer (0.6 molar, pH 6.0, 7.0 or 8.0) Incubated for 30 minutes at 60 ° and with 2.0 ml of aqueous sodium carbonate solution (15 mmol) diluted. The fluorescence is as in

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Example 1 measured.

Example 5

This example illustrates the influence of pH and incubation time on fluorescence:

Incub. LyS pH 6, Relative fluorescence intensity 0 Leu-ala Lys PH 7 , 0 Lys PH 8, D. Leu-ala Time - GIy - 740 GIy Leu-ala 740 GIy 1607 Min. - - - 800 745 IO96 620, 1216 1553 - - - - 757 930 1738 583 943 1457 15th 146 - 367 718 949 I996 5 ^ 5 893 1389 30th 157 52 470 520 936 2214 49O 840 1269 45 154 46 545 410 795 I98O 433 782 1146 60 127 47 553 248 651 I68O 370 726 915 90 113 39 548 198 507 II82 347 657 900 120 36 474 1044 620 i8o 240

The incubation and the fluorescence determination are carried out as described in Example 1. Lysine, glycine and leucylalanine are in equivalent amounts, 10 ιημΜοΙ per scoop, admitted.

Example 6

This example illustrates how fluorescence can do it occurs with normally occurring amino acids under different pH conditions. A typical dipeptide and ammonia are included in this example for comparison purposes.

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amino acid Rolative Fluorescence intensity at pH Hy pro 6.0 7.0. . 8.0 Asp O 0 0 G-Iu "Ό 39 95 Per 1 123 "■ 263 Try 2 2 18th VaI 3 24 34 Isoleu 4th 225 371 CySH 4th 263 399 Ala 4th . 27 30th Leu 4th 212 314; Tyr 6th 351 491 Thr 7th 282 516 Phe 8th 292 349 S he 8th 333 540 GIu (NH) 10 408 543 ' Mead 12 »,; 430 537: Asp (NH) 15th 523 629 Arg 15th 495 440 His 22nd . 275 455 - GIy 29 289 541 Cys 39 673 790 Lys 40 326 485 L-Leu-L-Ala 146 440 42? NH ₃ H 73 1825 1187 -: 0 0 0

The general rule given in Example 1 was:

ν ". ■■ - · ■ used to determine the relative fluorescence intensity,

where 10 m) | moles of each amino acid and an incubation time of 120 minutes at 60 ° were used for all pH values. . -

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' Example 7

This example illustrates the relative fluorescence intensity some biologically active peptides and amines

Peptides Relative
fluorescence Amines Relative
fluorescence insulin 246 Tyramine 176 Carnosine 219 Norepinephrine '62 Angiotensin 144 Normethanephriη 16I Glueagon 128 Doparnine 46 Bradykinin 104 3,4-dihydroxyphenyl
alanin 14th Vasopressin 69 Tryptamine 15th Oxytocin 14th Serotonin 18th
(128 *) Gastrin 0 histamine 142 Gramicidin 0 d-aminobutyric acid 96 Leucylalanine 365 Ethanolamine 86 Taurine 21Q Sperm 177 Cadaverine 91 Putrescine 97 Glucosamine 96 aniline Guanine 0 Adenine 0 Creatine 0 Guanidine 0

1 m) / mol, each compound was used and the fluorescence as described in the general procedure according to Example 1 developed at pH 7. For all measurements, a wavelength of 390 my was used for excitation and accordingly the emission at 480 m) l is determined.

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♦ Serotonin was incubated with the reagents at pH 6.

Example 8

This example illustrates the difference in sensitivity between the fluorescence determination method according to the present invention and the customary colorimetric determination method. '' ^f

absorption reagent Relative fluorescence reagent link minus minus read Blank sample. read Blank sample ner value 0.036 ner value 153 Glutamic acid 0.079 0.038 254 «15 Glycine 0.08l 0.040 916 803 Lysine 0.083 0.034 904 1355 Glycylglycine 0.077 0.049 1456 2017 Leucylalanine 0.092 0.032 2118 1027 Tyramine 0.075 0.027 II28 847 histamine 0.070 948 , - Blank sample 0.043 101

The same amounts (5 mJ (mol) of substrate are used for the determination according to the colorimetric method (Moore and Stein method) and the determination method according to the present invention at pH 7 as described in Example 1. From the above table it can be seen that with the colorimetric determination method the corrected absorption is of the same order of magnitude as the blank sample, which means that a high probability of error is introduced into the experiment; on the other hand, with the fluorometric determination method the corrected fluorescence generally differs by one order of magnitude compared to the blank sample, so here more precise analysis

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senresults are obtained and the possibility of determining smaller amounts of substrate is given.

Example 9

Different amounts of glutamic acid, glycine, leucylalanine and tyramine in Ιμΐ solvents are on paper or applied with silica gel loaded thin-layer plates. After developing with solvents and drying, the paper becomes or the plate with a solution of phenylacetaldehyde (15.InMoI) and Ninhydrin (5 mmol) sprayed in ethanol. The ethanol is allowed to evaporate and the chromatogram is then sprayed at room temperature with 0.2 molar phosphate buffer (pH 8.0). The moist chromatogram is then heated to 60 ° for 15 minutes and afterwards Cooling tested under a UV lamp (366 my). In the inventive Method, the detection limit for glutamic acid is 10 ~ mol; for glycine, leucylalanine and tyramine ΙΟ "Mol. In the usual ninhydrin parbo reaction, the Detection limit for all 4 named compounds at 10 ~ mol. ■

Example 10

10) | 1 of a standardized amino acid solution, each containing 1) iMol / ml of 20 common amino acids and ammonia, are applied to a 0.9 cm χ 55 cm column, which is coated with a resin (Beckman UR-30 resin for separation of acidic and neutral amino acids, 7 * 5 %> cross-linked, sulfonated, copolymeric styrene) is charged. The column is developed at a flow rate of 70 ml / hour first with 117 ml of 0.2 N sodium citrate (pH 3.25), then with 140 ml of 0.2 N sodium citrate (pH H, 3). Initially, the temperature is 45 ° and after 30 minutes over a period of 1 hour ■ - across increased to 55 °. For the rest, which the experiment:.: Ment takes, this temperature is maintained. The solution flowing out of the column is divided into two sample streams in a ratio of 9: 1: The larger of the two sample

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streams is passed through an amino acid analyzer (Beckman Model 120-C) in a conventional manner. The smaller sample stream, which flows at a flow rate of 0.1 ml / minute, is mixed with two further streams. One of these two streams flows at a rate of 0.3 ml / minute and consists of a solution obtained by mixing 2.67 g of ninhydrin, 180 mg of phenylacetaldehyde, 300 ml of ethanol and 150 ml of water. The other of the two streams, which flows at a rate of 2.0 ml / minute, consists of a 0.2 molar sodium phosphate buffer with a pH of 7.5. The corresponding flow rates are set and controlled by means of a metering pump. The total flow rate after mixing all the streams is 2.4 ml / minute. Every 1.5 seconds or so, an air bubble is created in the system to divide the flow into small segments. The flowing stream is passed through a short mixing spiral and then through a spiral that is immersed in a bath heated to 60 °. The total time it takes for the sample stream to travel through this heated coil is 30 minutes. After DurchfHessen the heated coil, the sample stream is cooled to 17 and through a 1 cm-Aminco half cell that passed is provided with a degassing _r. 75 % of the sample flow fHessen through this cell (speed 1.8 ml / minute) and 25 # are removed in the degassing device. The flow cell is look in an Aminco fluorescence microplate photometer with a 85 watt mercury vapor lamp General Electric, an Aminco No. 7.51 filters for the exciting radiation, an aperture set for maximum excitation, and an Aminco No. 4 filter is provided for detection. The fluorescence photometer is operated with a voltage setting of. kO, a sensitivity setting of 50% and a damping setting of 3% full scale (corresponding to 3 mVolt for full scale). The output of the fluorescence photometer is pounded with a Beckman 25 cm pen at a paper speed of 2.5 mm / minyte. ,

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For the usual colorimetric analysis, the recorder used in the Beckman amino acid analyzer is operated with a sensitivity setting of 5 mVolt full deflection and the usual 570 my output of the colorimeter is recorded. The paper feed speed is 2.5 mm / minute.

In a parallel experiment, a 0.9 cm χ 5 cm column is used which is charged with a Beckman · PA-35 resin for separating basic amino acids (8 ⁰ Jo cross-linked, sulfonated styrene copolymer). ■

The attached drawing shows the records of the * Writer under the test conditions described above gen. The first experiment shown in the drawing (left) corresponds to the experiment with the basic amino acids that were separated on a PA-35 column. The second in the The experiment shown in the drawing corresponds to the experiment with the neutral and acidic amino acids on a "UR-30 column separated '. - · ■ '-.,.

The test data shown in the drawing are summarized again in table form below. In this table the relative areas are reproduced under each of Schreiber aufgezeichneten'Kurve both the kolorimetrisehe d and for the fluorometric method and compared. These areas were corrected both with regard to the substrate concentration and with regard to the attenuation setting of the pen as they were used in the various experiments. The numbers appearing in the table represent the integrals of the measurement curves of each measuring device under the same conditions, based on the same amounts of each amino acid.

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amino acid Relative area under the maxima in Fluorescence photometry the drawing (corrected). ., 900 Colorimetry 750 OH-lysine 252 I35O Lysine 150 O Histidine 145 705 ammonia 49 430 ' Arginine 106 26O Cysteine 114 II50 Aspartic acid 145 296O Threonine 179 • 865 Serine 270 2370 Glutamic acid 190 755 Glycine 152 1500 Alanine 136 935 Cystine 161 1840 Valine 132 1090 Methionine 157 1760 Norleucine 145 .1750 Isoleucine • 125 1370 Leucine 144 1190 Tyrosine 170 2120 Phenylalanine ß-alanine 100

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Claims (1)

Claims ■ 1Λ method for determining. primary amino groups Containing * compounds in mixtures, characterized by that such a mixture with ilinbydrin and 'an arylalkylaldehyde the general formula where Ar is an aromatic radical and η is mean an integer from 1 to 8 » ■ ■ ■ - - ™ heated and the resulting fluorescent substance fluorometrically certainly* 2. The method according to claim 1, characterized in * that η in the formula of arylalkylaldehyde in claim 1 is a is an integer from 1 to 3. 3. The method according to claim 1 or 2, characterized in that the production of the fluorescent substance takes place in a solution * k. Method according to one of claims 1, 2 or 3, characterized in that phenylaeetalde-: hyd is used as the arylalkylaldehyde * 5 * method according to one of claims 1 to 3 or 4, characterized »that the pH of the solution is between 6 and 8 lies. 6. The method according to any one of claims 1 to 4 or 5, characterized in that the molar amounts of iiinhydrin and aryalkylaldehydeT compared to the molar amount of primary 209819/0925 Compound containing amino groups present in excess Sinti. 7. The method according to one of the claims 1 to 5 or 6, characterized in that containing primary amino groups Compound is a cf-amino acid « 8. The method according to any one of claims 1 to 6 or 7> characterized in that the primary amino groups contain ^ . the compound is a polypeptide " w 9 · Method according to claim 8, characterized in that that the polypeptide is selectively in a pH range of S _i; 0 to 7.0 is determined. -. - IQ .. Method according to one of claims 1 to 8 or %, characterized in that a fluorescence photometer is used for the fluorometric determination of the substance » 11. The method according to claim 3 *, characterized in that that the concentration of a primary amino group containing Compound in solution is quantified by "a) Determination of the fluorescence of a known concentration tion of said connection, b) Determination of the fluorescence of an unknown concentration at said connection, and c) Multiply the known concentration by the Ratio of the measured fluorescence values, the unknown and the known concentration ~. have delivered to said AmIn. 209819/0325 £ ί ^ 2U8603 - '■' · 12. * Method according to claim 1, characterized in that · that the primary amino group-containing compound is at least partially on the surface of paper or a for the thin layer chromatography suitable medium and that · ninhydrin and the arylalkylaldehyde on the surface of the mentioned medium suitable for the chromatography are brought. 13. The method according to claim 12, characterized in that that phenylacetaldehyde is used as arylalkylaldehyde. 14.- 'Method according to claim 12, characterized in that that the pH of the medium suitable for chromatography is kept between 6 and 8. 15. The method according to claim 12, characterized in that the molar amounts of ninhydrin and arylalkylaldehyde in the * Compared to the molar amount of the compound containing primary amino groups are present in excess. 16. The method according to any one of claims 12 to 14 or I5, characterized in that the arylalkylaldehyde in comparison j present in a three-fold molar excess to ninhydrin 17- method according to one of claims 12 to 15 or l6, characterized in that the compound containing primary amino groups is an ff-amino acid. l8. The method according to any one of claims 12 to 16 or 17, characterized in that the primary amine-containing groups Compound is a polypeptide. 9819 / 0.925 19 · Method according to claim 1 for determining a primary Compound containing amino groups by means of an automated device, characterized in that one a) a sample stream containing said primary amino groups containing compound in solution, continuously with a certain ratio of Treated ninhydrin and arylalkylaldehyde, b) the mixture obtained after step a) continuously passes through a heating zone to obtain a sample stream containing a fluorescent substance, c) the solution obtained according to step b) at constant speed through a flow cell of a Fluorescence photometer conducts, whereby fluorescent substance present is detected fluorometrically and is determined. 20. The method according to claim I9, characterized in that that the sample stream is at least part of a solution flowing out of a chromatography column. 21. The method according to claim I9 or 20, characterized in that that phenylacetaldehyde is used as arylalkylaldehyde. 22. The method according to any one of claims I9, 20 or 21, characterized in that the molar amounts of ninhydrin and phenylacetaldehyde compared to the molar amount of primary Compound containing amino groups are present in excess. 209819/0928 2U8603 23 · method according to one of claims I9 to 21 or 22, characterized in that the pH of the solution which is subjected to the fluorometric determination is between 6 and 8 is held. 24. The method according to any one of claims I9 to 22 or 23, characterized in that the compound containing primary amino groups is an ei amino acid. 25. The method according to any one of claims I9 to 23 or 24, characterized in that the compound containing primary amino groups is a polypeptide. 26. The method according to any one of claims I9 to 24 or 25 * characterized in that the total amount of a specific known, primary amino group-containing compound in one Sample flow is quantified by a) Determination of the total fluorescence of a known amount of said primary amino group-containing Link, b) Determination of the total fluorescence of an unknown amount of said primary amino groups containing Connection under the same conditions as in step a) and c) Multiplying the known amount by the ratio of the measured total fluorescence for the unknown and the known amount of said substance. 209819/0925 27. Automatic analyzer for carrying out the method according to any one of claims 1-26, characterized in that it in combination. . a) Devices for picking up a constant speed flowing sample stream, the primary one. Contains compounds containing amino groups, b) Devices for continuous feeding bestinsm - ',.-_ " ter amounts of reagents for the preparation of a ■ '' ".. fluorescent substance composed of primary amino groups ■ containing compounds to said sample stream, . 0) device for mixing said reagents with • said sample stream, .'ί ... ί d) Devices for heating the according to Schrifet c] @ r- ·. hold mixture,. ' • ■■■■) β) devices for fluorometrisehen ^ tuning of fluorescent material, which is in accordance with step ^; 'd) obtained mixture is located and f) Devices for determining · the contains. . - "'·. · ■ .'- 28. Analyzer according to claim 27, characterized in that «the feed device for the sample stream with. the outflow part of a device gur ohromatograpälschen ■ Separation of a mixture of · ρ3? 1ϋ} Μτ © compounds containing amino groups into the. 1 tion is connected . ' Ϊ9 / 092 Sf-'V £ <" '· ^{: 0} BiGiNAL INSPECTED 29. Reagent for the determination of compounds containing primary amino groups, characterized in that it is used as essential component is a solution of ninhydrin and an arylalkylaldehyde of the formula Ar (CH ₀ ) CHO
ε. η where Ar is an aromatic radical and η is a whole Number from 1 to 8, in an inert organic solvent. 30. Reagent according to claim 29, characterized in that that η in the formula of the arylalkylaldehyde in claim 29 is a is an integer from 1 to 3. 31. Reagent according to claim 29 or 30, characterized in that that the arylalkylaldehyde is phenylacetaldehyde. 32. Reagent according to claim 29, 30 or 31> characterized in that ninhydrin compared to phenylacetaldehyde ■ is present in a 10-fold molar excess. 33 · Reagent according to claim 29 to 31 or 32, characterized characterized that the phenylacetaldehyde compared to the ninhydrin in about a three-fold molar excess is present. 209819/0925 Empty se