این مسأله که اسیدآمینه های بالا در زمان های مختلف با نین هیدرین تشکیل رنگ می دهند، ارتباط مستقیم با ساختار اسیدآمینه دارد. در این فرایند امینواسیدها دو مولکول نین هیدرین را از طریق اتم نیتروژن به هم کوپل می کنند و خود آمینواسید به صورت یک آلدهید جدا می شود. برای این که آمینواسید بتواند به نین هیدرین اضافه شود، این کار را از طریق جفت الکترون آزاد نیتروژن آمین خود انجام می دهد. در مرحله بعد حذف یک مولکول CO₂ اتفاق می افتد و گروه R از طریق نیتروژن به نین هیدرین متصل می شود. به نظر می رسد که در این مرحله و نیز مرحله جداشدن آلدهید است که تفاوت اسیدهای آمینه در زمان رنگ دهی مشخص می شود. تمایل متفاوت گروه های هیدروکربنی موجود در اسیدآمینه های مختلف، سرعت حذف مولکول دی اکسید کربن را تحت تأثیر قرار می دهد. به فرایندهای زیر توجه کنید:

 

مشخصات نین هیدرین

physical propertis of nin hydrin

 Physical State: Crystalline powder 
Appearance: slightly yellow 
Odor: characteristic odor 
pH: Not available. 
Vapor Pressure: Negligible. 
Vapor Density: 6.16 (air=1) 
Evaporation Rate:Not applicable. 
Viscosity: Not available. 
Boiling Point: Not available. 
Freezing/Melting Point:466 deg F (dec) 
Autoignition Temperature: Not applicable. 
Flash Point: Not applicable. 
Decomposition Temperature:466 deg F 
NFPA Rating: (estimated) Health: 2; Flammability: 1; Reactivity: 0 
Explosion Limits, Lower:Not available. 
Upper: Not available. 
Solubility: Soluble in water. 
Specific Gravity/Density:0.86 
Molecular Formula:C9H6O4 
Molecular Weight:178.14

 

آزمایش نین هیدرین :

 

در این آزمایش ترکیبی به نام نین هیدرین با آمینواسید وارد واکنش می شود. در صورت برخورد و مجاورت این دو با یکدیگر ،گروه آمینی و کربوکسیل آمینواسید آزاد می شود . و خود آمینواسید به شکل به شکل یک ترکیب آلدهید CHO_R در می آید. و خود نین هیدرین به ماده ای به نام هیدرین دانتین  تبدیل می شود.

 

این واکنش برای هر اسیدآمینه ای پیش می رود. اما در اسیدآمینه پرولین تا حدی پیش می رود زیرا گروه متیل موجود در پرولین مانع از کامل شدن آزمایش می شود. به همین دلیل پرولین رنگ زرد ایجاد می کند.

 

یعنی  به طور خلاصه داریم :

 

نین هیدرین+ همه امینواسید ها= کمپلکس ارغوانی

 

 

 

 

 

 

 

 

 

 

 

Ninhydrin: Basic to Advanced

Pat A. Wertheim, Director of Training 
Forensic Identification Training Seminars, Ltd. 
1230 Hoyt St. S.E. 
Salem, OR 97302-2121 
1-888-235-1230

ABSTRACT: Ninhydrin, an amino acid reagent, may be applied to porous surfaces in a variety of solutions to develop latent finger and palm prints. The choice of application depends on the surface being processed, the expertise of the examiner, and the equipment and supplies available. Several chemical formulas are discussed, along with methods of application. Choosing the correct formula and method in any given situation will maximize the chances of success while minimizing the hazards, both to the examiner and the evidence.

GENERAL CONSIDERATIONS

Ninhydrin (synonym: 1,2,3-triketo-hydrindene hydrate) has been recognized since around the turn of the century as a reagent for detecting amino acids. However, it was not until the mid 1950's that it was discovered ninhydrin could be used to develop latent prints on porous surfaces. When ninhydrin comes into contact withamino acids in fingerprint residue, the reaction yields a red to purple print.

While effective on virtually any porous surface, ninhydrin is most appropriate for paper. On wood, silver nitrate may yield prints with darker ridges and better contrast because sometimes faint purple ridges of a ninhydrin print may get obscured by the wood grain and brown background color. This discussion, therefore, will address the use of ninhydrin on paper.

Several variables come into play. First, the types of amino acids and their concentration in the print determine whether a reaction will occur. Second, the reaction is most effective in an acidic environment but many white papers have calcium carbonate fillers or whiteners. Such fillers result in a basic environment tin the paper unless a mild acid is added to the ninhydrin reagent. Third, temperature and humidity influence the development of prints. Fourth, the strength of the ninhydrin and the type of carrier affect the reaction. With all of these variables, the development time and the darkness of the print may differ widely from case to case. it is always suggested that sample prints be placed on paper similar to that of the evidence processed as a control using the ninhydrin solution before actual evidence items are processed. Once the efficacy of the reagent solution has been established, the examiner may proceed with the evidence itself. Experience has shown that occasionally a latent print may only appear after several applications of ninhydrin.

If more than one method of chemical processing is to be used, the methods must be used in the correct order to avoid ruining the chances of subsequent success. On paper, the various methods should be used in the following sequence: 1) iodine fuming, 2) DFO (followed by florescence examination,) 3) ninhydrin, and 4) silver nitrate or physical developer. Not all methods may be advisable in all cases. Depending on the priority of the case and the resources available, an examiner may choose to use only one or two of these methods. But anytime a combination is chosen, the methods should be used in the correct sequence.

In many cases where fingerprints would be important, handwriting is also present on the paper. It should be recognized that any liquid soaking into the paper will alter the indentations left by the writing instrument. In addition, most chemicals used to dissolve ninhydrin also dissolve inks to some degree. Therefore, document examination should always precede fingerprint examination.

SAFETY CONCERNS

Many of the solvents used for ninhydrin are toxic and flammable, while even dry ninhydrin itself is harmful. Therefore, adequate safety precautions must be taken. Nitrile gloves should be worn rather than latex, as latex is soluble in many of the reagents but nitrile is resistant to chemicals. Goggles and a breathing mask should also be used. A lab coat should be worn o protect clothing. For safety reason, dipping the item into the solution or painting the solution onto the item with a cotton ball are preferable to spraying the solution onto the paper. because many of the solvents used are flammable, adequate ventilation should always be used and it should be ascertained that no source of flame or spark is present which might ignite fumes. A vent hood is recommended to remove fumes from the lab, both to remove any chance of explosion, and because many of the solvents and the ninhydrin itself may be hazardous.

PREMIXED SPRAYS

Several premixed sprays are available, including both aerosol cans and pump sprayers. Premixed sprays may be the most convenient way to purchase ninhydrin. However, it is much less expensive to mix your own solutions. And while all of these premixed sprays work well when "fresh," their shelf life is measured in months, not years. Because the age of a container may not be known when purchased, and the conditions of storage and shipment are variable, there may be little uniformity from can to can. In addition, contamination may occasionally render a can of premixed spray not only ineffective, but damaging to the item being sprayed. Finally, spraying is no longer recommended as a method of application because of the excessive amount of both ninhydrin and carrier introduced into the air. Even when working under a vent hood, airborne droplets of solution may find their way back into the laboratory atmosphere.

MIXING YOUR OWN SOLUTIONS

While mixing your own solutions may not be quite as easy as using a spray can off the shelf, it is significantly less expensive and generally more effective for developing latent prints. A number of different solutions may be prepared, depending on the specific application involved and your comfort in mixing chemicals. Different solutions have different characteristics, as well as different advantages and disadvantages.

Solutions may be mixed ahead and stored for later use, or a small amount may be mixed sufficient only for the application at hand. Some solutions may require a "stock solution" and a "working solution." In those, a concentrated solution is prepared (the stock solution) and mixed with a carrier prior to use (the working solution.) You may select as simple or sophisticated a solution as you are comfortable with, and either mix it ahead or for each use separately. Since it is up to you, the examiner, to determine what is most appropriate and defend your choice in court, it is important that you be familiar with different methods and make an informed decision on which one to use. Two ninhydrin solutions are presented here for your consideration, one simple to prepare, the other slightly more sophisticated.

SIMPLE SOLUTION: NINHYDRIN IN ACETONE (or methanol, or ethanol)

The simplest solution to prepare is made by using a 25 gram jar of ninhydrin powder or crystals. Obtain a gallon (4 liters will do) can or jar of acetone from a paint store or chemical supply house. Pour the entire jar of ninhydrin powder into the gallon container and stir until completely dissolved. The solution is ready for use and should last a month or two if kept out of light and away from heat.

Because of shelf life concerns, unless you use a large amount of ninhydrin, it is not recommended you mix it all at once. Instead, by using approximately the proportions given above, smaller amounts of solution may be prepared. For example, for a single use amount for a forged check. a few spoons of acetone may be used, and a "pinch" of ninhydrin stirred in until dissolved. WARNING: Acetone dissolves most inks, and the writing on the check, threatening letter, or drug ledger may be completely washed out using this simple method.

based on availability, methanol (methyl alcohol) or ethanol (ethyl alcohol may be preferred to acetone. Alcohols evaporate more slowly than acetone, and therefore the paper takes longer to dry. Also, the alcohols do not have as strong an odor os acetone, and one is less apt to be aware of breathing hazardous fumes. Like acetone, alcohols dissolve ink. Acetone and alcohols are flammable, so care must be exercised in keeping these solutions away from any source of ignition.

BETTER SOLUTION: NINHYDRIN IN HEPTANE

With the banning of freon, it is no longer possible to use of the most popular and effective solutions for ninhydrin. An alternate carrier has been sought for several years. The Police Scientific Development Branch of the Home Office, UK, recently found a suitable replacement formula using heptane. Their formula for ninhydrin in heptane is as follows:

Ninhydrin....................................5 grams 
Ethanol.....................................75 milliliters 
Ethyl Acetate...............................25 milliliters 
Acetic Acid..................................3 milliliters 
Heptane......................................1 liter

First, dissolve the ninhydrin in the ethanol, ethyl acetate, and acetic acid. Then add the heptane. The solution has a shelf life of several months. Heptane is flammable, so care must be exercised in keeping the solution and fumes away from any source of ignition. This solution results in only minimal running of ink and is very effective at developing latent prints. In fact, efficacy studies at the Home Office indicate this formula may even be more effective than the old freon formula.

CONCLUSION

When fingerprinting paper items, freshly mixed solutions are generally more dependable than premixed chemicals. if a document examination is appropriate, it should be done before fingerprint chemicals are applied. If more than one technique is to be tried, such as iodine or silver nitrate, the various techniques must be done in the correct sequence. From a safety standpoint, dipping the item in a tray of ninhydrin solution or painting the ninhydrin onto the surface with a cotton ball are preferable to spraying. Acetone or alcohol solutions are more likely to damage inks than a heptane formula. Nitrile gloves, a lab coat, safety goggles, and breathing mask should be worn at all times when working with ninhydrin powder or solutions.

 

2,2-Dihydroxyindane-1,3-dione

Identifiers

CAS number 485-47-2 

PubChem 10236

ChemSpider 9819 

ChEMBL CHEMBL1221925 

Jmol-3D images Image 1

SMILES

[show]

InChI

[show]

Properties

Molecular formula C9H6O4

Molar mass 178.14 g mol−1

Appearance White solid

Density 0.862 g/cm3

Melting point

250 °C (decomp.)

Hazards

MSDS External MSDS

R-phrases R22, R36, R37, R38

S-phrases S26, S28, S36

  (verify) (what is: /?)

Except where noted otherwise, data are given for materials in their standard state (at 25 °C, 100 kPa)

Infobox references

Ninhydrin (2,2-Dihydroxyindane-1,3-dione) is a chemical used to detect ammonia or primary and secondary amines. When reacting with these free amines, a deep blue or purple color known as Ruhemann's purple is produced. Ninhydrin is most commonly used to detect fingerprints, as the terminal amines of lysine residues in peptides and proteins sloughed off in fingerprints react with ninhydrin.[1] It is a white solid which is soluble in ethanol and acetone at room temperature. [2] Ninhydrin can be considered as the hydrate of indane-1,2,3-trione.

Contents  [hide] 

1 Uses

2 Reactivity

3 See also

4 References

[edit]Uses

Ninhydrin can also be used to monitor deprotection in solid phase peptide synthesis (Kaiser Test).[3] The chain is linked via its C-terminus to the solid support, with the N-terminus extending off it. When that nitrogen is deprotected, a ninhydrin test yields blue. Amino-acid residues are attached with their N-terminus protected, so if the next residue has been successfully coupled onto the chain, the test gives a colorless or yellow result.

Ninhydrin is also used in amino acid analysis of proteins: Most of the amino acids are hydrolyzed and reacted with ninhydrin except proline; Also, certain amino acid chains are degraded. Therefore, separate analysis is required for identifying such amino acids that either react differently or do not react at all with ninhydrin. The rest of the amino acids are then quantified colorimetrically after separation by chromatography.

A solution suspected of containing the ammonium ion can be tested by ninhydrin by dotting it onto a solid support (such as silica gel); treatment with ninhydrin should result in a dramatic purple color if the solution contains this species. In the analysis of a chemical reaction by thin layer chromatography (TLC), the reagent can also be used. It will detect, on the TLC plate, virtually all amines, carbamates and also, after vigorous heating, amides.

When ninhydrin reacts with amino acids, the reaction also releases CO2. The carbon in this CO2 originates from the carboxyl carbon of the amino acid. This reaction has been used to release the carboxyl carbons of bone collagen from ancient bones[4] for stable isotope analysis in order to help reconstruct the palaeodiet of cave bears.[5] Release of the carboxyl carbon (via ninhydrin) from amino acids recovered from soil that has been treated with a labeled substrate demonstrates assimilation of that substrate into microbial protein.[6] This approach was successfully used to reveal that some ammonium oxidizing bacteria, also called nitrifying bacteria use urea as a carbon source in soil.[7]

A ninhydrin solution is commonly used by forensic investigators in the analysis of latent fingerprints on porous surfaces such as paper. Amino acid containing fingermarks, formed by minute sweat secretions which gather on the finger's unique ridges, are treated with the ninhydrin solution which turns the amino acid finger ridge patterns purple and therefore visible.[8]

[edit]Reactivity

Indane-1,2,3-trione

IUPAC name[hide]

Indane-1,2,3-trione

Other names[hide]

Indanetrione

Identifiers

CAS number 938-24-9

PubChem 70309

Jmol-3D images Image 1

SMILES

[show]

Properties

Molecular formula C9H4O3

Molar mass 160.13 g mol−1

Appearance white powder

Density 1.482 g/cm3

Boiling point

338.4 °C, 612 K, 641 °F

Except where noted otherwise, data are given for materials in their standard state (at 25 °C, 100 kPa)

Infobox references

The carbon atom of a carbonyl bears a partial positive charge enhanced by neighboring electron withdrawing groups like carbonyl itself. So the central carbon of a 1,2,3-tricarbonyl compound is much more electrophilic than one in a simple ketone. Thus indane-1,2,3-trione reacts readily with nucleophiles, including water. Whereas for most carbonyl compounds, a carbonyl form is more stable than a product of water addition (hydrate), ninhydrin forms a stable hydrate of the central carbon because of the destabilizing effect of the adjacent carbonyl groups.

Note that in order to generate the ninhydrin chromophore, the amine is condensed with a molecule of ninhydrin to give a Schiff base. Thus only ammonia and primary amines can proceed past this step. At this step, there must also be an alpha proton (H* in the diagram) for Schiff base transfer, so an amine adjacent to a tertiary carbon cannot be detected by the ninhydrin test. The reaction of ninhydrin with secondary amines gives an iminium salt, which is also coloured, and this is generally yellow-orange in color.