Amorphous deposits

Amorphous material can be loosely defined as particles that, under magnification, have no specific shape or morphology. The common types of deposits that can be found in wine, and fall into this category are:

Combinations of the above are also possible, such as complexes between proteins and phenolics, proteins and polysaccharides and polysaccharides and tannins. It is impossible to make a positive diagnosis of an amorphous haze or deposit based on microscopy alone. The first step in identifying an unknown amorphous deposit is to test its solubility. Shown here are some simple solubility tests to help decide what tests to carry out.

Solubility tests for amorphous deposits

Reagents :

  1. 0.1M sodium hydroxide
  2. Hydrochloric acid (ca. 10%) – dilute one part concentrated hydrochloric acid (ca. 32%) in two parts cold distilled water.
  3. 50% v/v ethanol>

Examine the solubility of small portions of centrifuged deposit in the sodium hydroxide and hydrochloric acid solutions. Solubility in sodium hydroxide is typical of proteinaceous deposits, and also some phenolic type instabilities. Solubility in hydrochloric acid suggests metallic instabilities or colouring matter (Anon 1984). Solubility in 50% ethanol indicates the presence of polyphenolics (pigments and tannins).


Protein hazes are the result of the wine containing heat-unstable grape protein, which slowly denatures and precipitates if the wine is warmed, or forms insoluble complexes with polysaccharides or polyphenolics.

If the isolated deposit is freely soluble in 0.1M sodium hydroxide, protein is most likely present. To confirm the presence of protein in a deposit the following test is performed:

Nigrosine test for protein

Reagents :

  1. 0.1M sodium hydroxide
  2. Staining solution – Dissolve 125 mg of nigrosine (BDH) in a solution comprised of 483 mL methanol, 86 mL of glacial acetic acid, and 431 mL of distilled water.
  3. Destaining solution – Mix 50 mL of methanol, 10 mL of acetic acid, and 50 mL of distilled water.

Dissolve a portion of the deposit in 0.1 M sodium hydroxide (approximately 1-2 mL), and then apply one or two drops of the resulting solution to a filter paper. Evaporate off the liquid using a blow-drier, apply a further one or two drops of solution, and re-evaporate with the blow-drier. More or less of the solution may be required, depending on how much material is present. Place the filter paper in a small petri dish, and flood with the nigrosine staining solution. Pour off the solution after about 15 minutes (the solution can be re-used), and cover the filter paper in the destaining solution, until the filter paper is almost white. Proteinaceous material will be stained a bluish-grey colour (Anon. 1984). [Click here to see a video demonstration: Part I | Part II]

Infra-red spectroscopy can also be used to identify a protein deposit. Protein deposits can be prevented from forming in wines by fining with bentonite at a rate sufficient to render the wine heat stable. The heat stability of a wine is tested using the following method:

Test for heat stability of a wine

Filter two 20 mL samples of the wine through a 0.45 m membrane into two 25 mL screw-capped test tubes. Examine both samples using a strong light source to ensure that the wine is brilliantly clear after the filtration. Place one of the tubes in a water bath pre-heated to 80°C, ensuring that the entire volume of wine in the tube is immersed in water. Leave the other tube (the control sample) at approximately 20°C. Heat the samples at 80°C for two hours (minimum) and up to six hours. While six hours has been typical industry practice for the heat test, it’s important to note that two hours of heating time has been shown to be sufficient (Pocock and Waters 2006). After heating, remove the heated tube from the water bath and cool under running water or by leaving on the bench for at least two hours. A minimum of two hours cooling is required for the heated proteins to aggregate and precipitate, so chilling on ice to quickly lower temperature is not recommended.

After the cooling period, examine the heated sample for any haze by holding it against the strong light source – then compare against the unheated control. If a haze is observed in the heated sample that is not present in the unheated control, the wine is considered to be heat unstable and therefore susceptible to the formation of protein deposits. If the heated wine remains clear, the wine is heat stable and therefore not likely to form a protein haze or deposit.

Because the observation of the haziness of the heated sample is subjective, sometimes conflicting results can arise between different laboratories or different analysts. To avoid this, it is best to carry out the test in the most stringent way possible. This can be achieved by using a focusable light equipped with an adjustable iris diaphragm, which produces a narrow and focused beam of light, allowing very faint hazes to be detected. If the beam of light is only visible as it enters and leaves the tube, the sample passes (i.e. is heat stable) – but if the beam can be seen through the sample, it fails and the wine is considered heat unstable.

A turbidimeter can be used for more objective comparison of the turbidity in the two samples. In this case, wines that exhibit a turbidity increase of greater than a given criterion for nephelometer turbidity units (NTU) after heating, as compared with the unheated control, can be considered to have failed the heat stability test. Some laboratories use a criterion of 0.5 NTU, but other practitioners in industry have indicated that a more reasonable criterion is 2.0 NTU (Wilkes, personal communication 2004).


  • Pocock, K.F., Waters, E.J. 2006. Protein haze in bottled white wines: how well do stability tests and bentonite fining trials predict haze formation during storage and transport? Aust. J. Grape Wine Res. 12(3) 212-213.
  • Wilkes, E. 2004. Group Manager – Commercial Services, AWRI, personal communication.

Once a positive identification of protein is made it may be necessary to check for the presence of copper if bright, shiny-looking spheres were observed during examination of the deposit by phase contrast microscopy (see Metal haze section). Presence of copper will indicate a copper/protein ‘casse’.


Polysaccharides originate from the grape (such as pectins) and from Botrytis infection, are released by yeast during fermentation and during lees contact, and are also released by some bacteria. Polysaccharides can form colloidal hazes in wines which make clarification and filtration difficult. Since polysaccharides form gelatinous aggregates when mixed with alcohol solutions, the simple test described below may be used to determine if polysaccharides are present in a haze.

Alcohol precipitation test for polysaccharides

To a test tube containing 10 mL of wine, add 5 mL of 96% v/v ethanol and mix thoroughly. The formation of white filaments is indicative of the presence of polysaccharides.

If filaments do not form, but a haze develops upon mixing, the following more sensitive test may be performed.

After mixing the 10 mL of wine with 10 mL of 96% v/v ethanol, allow the mixture to stand for 30 minutes and then centrifuge, decant and discard the supernatant. Redissolve the deposit in 2 mL of water and add 1 mL of 96% v/v ethanol. The formation of filaments is indicative of polysaccharides.


Unstable caramel is one of the main causes of instability in brandy, and sometimes other spirits and wines to which it has been added for colour adjustment. Caramel can also form a deposit in sweet wines which have been pasteurised. The main causes of caramel colour instability, leading to haze and deposit formation, are as follows:

  1. Use of the wrong type of caramel (the winemaker should use wine caramel for wines and spirit caramel for brandy).
  2. Use of old caramel. The maximum shelf life of caramel kept under cool conditions is one to two years.
  3. Mixing two different makes or types of caramels together, or blending products coloured with different caramels.
  4. Reaction with wine or brandy components such as:
    1. Protein, including excess protein from proteinaceous fining agents
    2. Calcium
    3. Iron
    4. Tannins and wood extractives.

Deposits that contain caramel are usually brown to black in colour. If caramel is suspected in a deposit the following test for carbohydrates (di- and polysaccharides) is recommended.

Spot test for the presence of carbohydrates (di- and polysaccharides) in a solid material

Reagents :

  1. 10% solution of aniline in 10% acetic acid
  2. Phosphoric acid

Isolate the material in a microcrucible. Add a drop of phosphoric acid, and then cover the crucible with a disk of filter paper which has been moistened with the aniline acetate reagent (reagent 1, above). A small watch glass is then placed on top, as a paper weight.

The crucible is then gently heated with a small Bunsen flame for about 30 seconds, avoiding spattering. A pink to red colour appears, the shade depending on the quantity of carbohydrate present (ie, the darker the shade, the higher quantity of carbohydrate present). This colouration will fade after about five minutes.

It is recommended that positive and negative controls (e.g. sucrose and sodium chloride, respectively) be tested at the same time as the unknowns, and that the heating period be consistent for all samples tested.

The test, described by Feigl (1956), is based on the cleavage of di- and polysaccharides to furfural or its derivatives, which then form a coloured ‘Schiff base’ with aniline acetate.

Red Pigments

Most amorphous deposits found in red wine, in particular, are coloured. The compounds responsible for the colour in red wines are a class of flavonoid phenolic compounds known as anthocyanins. A simple test for red pigments, based on solubility in ‘LA’ reagent is described below.

Test for red pigments based on solubility in ‘LA’ reagent

Reagent :

  1. ‘LA’ reagent – add 25 mL of concentrated hydrochloric acid to 500 mL of n-butanol.

Add approximately 2 mL of ‘LA’ reagent to a small portion of deposit in a test tube or centrifuge tube, and then place in boiling water for about 15 minutes. Solubility and production of a dark red colour indicates the presence of red pigments.


Phenolic compounds in wine can, over time, condense with other phenolic compounds and thus polymerise. When the polymer reaches a certain size it is no longer soluble in the wine medium, and thus precipitates out. Polymers which have a chain length of greater than four units are known as tannins. In solution the tannins can interact with proteins and mutually precipitate. Tannins are classified into two categories:

  1. Hydrolysable Tannins – copolymers of gallic/ellagic acids with sugar
  2. Condensed Tannins – polymers of flavanoids which are condensed mainly through carbon-carbon bonds.

Following are the confirmatory tests for polyphenolics and condensed tannins:

Test for polyphenols and tannins

Transfer a portion of the deposit onto a watch glass. Add a drop of Folin-Ciocalteu reagent (available from chemical suppliers) diluted 1:10 with distilled water.

Phenolic complexes dissolve or partially dissolve and form a grey-blue to dark-blue solution.


Cyanidin test for condensed tannins

Reagents :

  1. Methanol
  2. Hydrochloric acid (ca. 32% v/v)

Wash a portion of the deposit in 2 x ca. 1 mL of methanol, re-centrifuge, then add 1 mL of methanol and 1 mL of hydrochloric acid (ca. 32% v/v) and heat at 80°C for 30 minutes. The formation of a pink-red colour indicates the presence of condensed tannins. The test conditions result in hydrolysis of any tannins which may be present to cyanidin, which has a reddish colour (Porter et al. 1986).

Metal Hazes

The main metals that can cause instabilities in wine are copper, iron and occasionally aluminium. Other metals present in wine, including tin, lead and silver, may be capable of causing instability in wine, however, these metals are unlikely to be present in any more than trace amounts in wine.

  • More information about these less common metal instabilities is found here.


Copper instability is the most common metal related instability, largely because of the small concentrations of copper required to cause instability, if certain other factors are also present.

Although higher concentrations are sometimes observed, wines typically contain approximately 0.3 mg/L of copper or less (current Australian food laws specify a maximum of 5 mg/kg for ‘beverages and other liquid foods’). The Institute has in the past suggested that 0.5 mg/L is a reasonable maximum ‘safe level’ in order to avoid copper instabilities, but instabilities have been observed in wines containing less than this amount of copper. Stability depends not only on copper concentration but on several other aspects of wine composition and storage conditions.

Copper instability, or ‘copper casse’, as it is often called, is limited largely to white wine and develops some time after bottling. The instability is observed as a fine haze initially, which may subsequently form an off-white to mid brown deposit. Copper casse usually appears as amorphous granular particles when examined by phase-contrast microscopy.

Copper is a micronutrient which is required in trace quantities by the vine as a component of certain oxidative enzymes. Its presence in wine, over and above the trace levels which are incorporated into the grape berry by translocation from soil, is likely to have originated from the following sources:

  1. Additions of copper sulfate (CuSO4.5H2O), made to correct perceived sulfide faults, probably are responsible for the majority of copper instability problems. Accidental or incorrectly calculated additions of copper sulfate are also quite common.
  2. Residues from copper-based vineyard sprays such as Bordeaux Mixture (containing copper sulfate) or copper oxychloride can lead to extremely high copper levels in juice if sprayed incorrectly or if minimum withholding periods are not observed. However, the copper tends to bind to and co-precipitate with the yeast lees, and the concentration of copper usually drops to about 1.0 mg/L following fermentation.
  3. Contact between wine and unprotected surfaces of copper or copper alloys such as brass (copper + zinc) or bronze (copper + ~ one third tin). It is likely that use of these types of materials has persisted in wineries for so long partly because of their tendency to remove hydrogen sulfide by precipitation as copper sulfide. Brass fittings are also a recognised source of lead contamination in wines.
  4. Fining agents (apart from copper sulfate) have also been suggested as causes of copper contamination in wines.
  5. Unwashed filter pads may be a source of copper, as well as iron and calcium.

Further detailed technical information about formation and prevention of copper casse is found here.

Another possible source of copper contamination is filter pads that have been rinsed with hot water that has been contaminated from copper pipes.



Instabilities due to iron are much less frequently encountered than instabilities due to copper. Australian wines generally contain about 0.5 to 5.0 mg/L of iron, with the majority containing less than about 2.0 mg/L. There is no limit set by the National Food Authority for iron in wine, but like copper, there is a range of concentrations suggested to avoid instability, most suggesting about 5 to 6 mg/L as a ‘maximum safe level’. Iron can impart a metallic taste if present at 10 mg/L or higher.

In white wines, iron (III) reacts with phosphate anions to form ferric phosphate, which precipitates out to form a white haze and/or precipitate. Because of this it is sometimes referred to as white casse. An increase in the concentration of phosphate ion would therefore be expected to increase the likelihood of instability, assuming that other conditions favouring instability were also present. Diammonium phosphate additions are the most likely way in which the concentration of phosphate ion can be increased.

In red wines, iron (III) reacts with the tannate ion to form a bluish black deposit of ferric tannate, which is sometimes referred to as blue casse. This can also occur in fuller-bodied white wines, such as those made with skin contact or containing heavy pressings.

Sources of iron in wine are summarised below:

  1. Contact of juice or wine with unprotected mild steel or cast iron surfaces. Epoxy or wax coated mild steel tanks should be checked annually, and resurfaced as necessary.
  2. Contamination from fining agents, particularly bentonite. The Institute’s recent bentonites survey found that three of eight commercially available bentonites exceeded the iron limit specified by the OIV for bentonite. One supplier actually has a bentonite available which is promoted on the basis of its low iron content – called Eisenarm (iron-poor).
  3. Contamination from unwashed filter pads

More detailed technical information about iron instabilities is found here.

A hazy wine may be subjected to the following clarification tests to determine whether the haze is copper or iron related.

Clarification test for the presence of copper and iron

To approximately 20 mL of wine contained in a test tube, add 5 drops of 30% hydrogen peroxide.

If the haze clears, the presence of copper is suspected. If the wine becomes more cloudy, the presence of iron is suspected.

To approximately 20 mL of wine contained in a test tube, add several drops of a 0.1% sodium sulfite solution.

If the haze clears, the presence of iron is suspected. If the wine becomes more cloudy immediately, the presence of copper is suspected.

If sufficient deposit or material responsible for the haze can be isolated, the simple ferrocyanide/ferricyanide test for copper and iron can be performed.

Ferrocyanide/ferricyanide test for metals

Reagents :

  1. Hydrochloric acid (ca. 10%) – dilute one part concentrated hydrochloric acid (ca. 32%) in two parts cold water.
  2. Mixed ferrocyanide/ferricyanide solution – dissolve 5 g of each of potassium ferrocyanide and potassium ferricyanide in distilled water, mix together, and make to 100 mL with distilled water (CARE!!).

Dissolve a small portion of deposit in approximately 2 mL of 10% hydrochloric acid, and then add a couple of drops of mixed ferrocyanide/ferricyanide solution. A blue or green colour indicates the presence of iron, a reddish brown colour indicates the presence of copper (a white deposit indicates the presence of zinc).


Confirmatory test for the presence of copper

Dissolve the deposit in a few mL of 25% hydrochloric acid and add concentrated ammonium hydroxide drop by drop.

A blue colour indicates the presence of copper.

If the isolated deposit (which was soluble in 10% hydrochloric acid) was somewhat soluble in 0.1M sodium hydroxide when subjected to the Solubility tests for amorphous deposits, then protein may also be present. The presence of protein can be confirmed using the Nigrosine test for protein. If the confirmatory tests indicate the deposit contains copper, and a positive result is obtained for the Nigrosine test, then the deposit is a copper/protein ‘casse’.

If the deposit was isolated from a white wine, formed a white haze or precipitate, and the confirmatory tests indicate the deposit contains iron, then the deposit is likely to be iron phosphate.

Test for phosphate using ammonium molybdate./benzidine test (Feigl 1958 – spot tests. In: Inorganic analysis 5th edition, pp333-334)

If the deposit was isolated from a red wine, formed a bluish black deposit, and the confirmatory tests indicate the deposit contains iron, then the deposit is likely an iron-tannin complex. The test for polyphenols and tannins combined with the Cyanidin test for condensed tannins would confirm the presence of tannins in such a complex.

AAS Methods:

  • Dissolving deposit in NaOH or HCl then analyse by AAS
  • Concentration of Cu and Fe in wine by AAS