Once a deposit has been categorised as crystalline, it should be washed with cold water or dilute (10% v/v) ethanol, recentrifuged and dried, then steps can be taken to identify exactly what crystalline compound it is. Infra-red (IR) spectroscopy is probably the best method to achieve this. A spectrum of the deposit is obtained, using a method appropriate to the spectrometer used, and then compared with reference spectra of likely crystalline compounds. Crystalline compounds often produce detailed and therefore diagnostic spectra, making this an excellent technique for their identification. If IR spectroscopy is not available, it is more difficult to obtain a definite identification, but there are tests which can be used to narrow down the possibilities.
Some crystalline deposits found in wine and a description of their usual crystal shapes are listed below:
- Potassium hydrogen tartrate– ‘boat’ or lens shaped
- Calcium tartrate– rhomboid or prism shaped
- Ellagic acid– needle shaped
- Calcium oxalate– needle shaped, cubic
- Calcium sulfate– needle shaped
- Flavonols– needle shaped
- Calcium mucate– white clumps, no defined crystal shape
- Quercetin dihydrate – thin needle shaped, sometimes yellow in colour
KHT is the most common crystalline deposit found in wines. It occurs when bitartrate ions (HT-), formed by the dissociation of tartaric acid, precipitate out with potassium ions. The characteristic boat or lens-shaped morphology of these crystals is shown here. However, occasionally KHT adapt other morphologies, some of which are shown here. More detailed technical information about KHT deposits is found here. If IR spectroscopy is not available to identify a suspected KHT deposit, it can be identified by carrying out the sodium metavanadate test for tartrates combined with a flame test for the presence of potassium.
|Sodium metavanadate test for presence of tartrate
Dissolve a small quantity of the crystalline deposit in the dilute sulfuric acid. Add a few drops of the sodium metavanadate solution. A yellow-orange colour indicates the presence of tartrate (Zoecklein et al. 1990). A control sample of KHT or CaT can be tested at the same time as the unknown deposit, for comparative purposes.
|Flame test for presence of potassium and calcium in crystalline deposits
Using a stainless steel spatula or a short length of nichrome wire, place a small quantity of crystals in a Bunsen flame. Potassium hydrogen tartrate will burn with a violet-coloured flame (Anon. 1984) and leave behind solid black residue. Calcium tartrate (and other calcium salts) will produce a brick red-coloured flame (Anon. 1984) and leaves behind a white ‘fluffy’ residue.Both tartaric acid and potassium are natural components of grapes and wine, so precipitation of KHT is one of the normal steps in the wine making process. It only becomes a problem if the deposit forms in the bottle, where it is considered an aesthetic defect, even though it has no detrimental effect on wine quality. Prevention of KHT precipitation in bottle is usually achieved by thorough cold stabilisation of the wine before bottling. A predictive test for cold stability should be applied to the wine before bottling. There are several commonly used methods for determining cold stability, however, the Institute recommends the following refrigeration method, since it relates well to the actual deposition of crystals in wine over time.
|Test for tartrate stability
Filter approximately 150 mL of the wine to be tested through a 0.45 µm membrane, and place in a 200 mL screw-capped glass bottle. Place the sample in a –4°C liquid bath or a freezer set to –4°C. After a period of three days, remove the sample from the bath and check immediately for the presence of crystals, with the aid of a bright light.The wine is interpreted as being cold stable if no crystalline deposits are observed.Some red wines may develop deposits of phenolic compounds during the test. These will usually redissolve when the sample is returned to room temperature, unlike tartrate crystals which will persist. The presence of these phenolic deposits therefore does not indicate tartrate instability.If you are unable to carry out this test yourself you can send a sample to AWRI Commercial Services.
Calcium tartrate is another fairly common crystalline deposit found in wine, formed from the calcium and tartaric acid which both occur naturally in grapes and wines. These crystals are usually described as rhomboid or prism-shaped (see photo). In the absence of IR spectroscopy, a suspected CaT deposit can be identified using the sodium metavanadate test for tartrates, the flame test for calcium discussed above, and the test for calcium described below. While CaT deposits are less common than KHT, they are also harder to predict or prevent. CaT precipitation is much slower than that of KHT and is temperature independent, which means that cold stabilisation is not an effective method for preventing this type of instability. While calcium occurs naturally in grapes and wines, levels can be elevated by the use of calcium salts as additives or fining agents or by storage of the wine in concrete tanks. It has been suggested that wines containing 80 mg/L or more of calcium are at risk of CaT precipitation.
- More detailed technical information on CaT deposits is found here.
|Test for calcium
Add a small amount of oxalic acid (spatula tip) to approximately 10 mL of centrifuged wine. Crystal formation is indicative of the presence of calcium. To confirm this result, add several drops of concentrated sulfuric acid to dissolve the precipitate. The addition of excess methanol and heat will cause the precipitate (calcium sulfate) to reappear.
|Predictive test for CaT stability
See page 542 The Tech. of Winemaking, Amerine et al 1980. (CP etc)
Ellagic acid is a crystalline deposit found fairly rarely in wine. Its presence is normally associated with treatment of wines with oak chips or shavings. Such treatment results in the rapid extraction of certain hydrolysable tannins, including ellagitannins, from the oak. These slowly hydrolyse to form crystals of ellagic acid, which have very low solubility in wine and therefore precipitate out. This precipitation may occur in the bottle if insufficient time is allowed for hydrolysis of the ellagitannin precursors before bottling. The rate of hydrolysis is dependent on several factors, including wine storage temperature. A period of at least four weeks is generally recommended between treatment with oak chips or shavings and bottling, in order to allow hydrolysis and precipitation to occur before bottling (Pocock, Strauss and Somers 1984).
Ellagic acid crystals are identified using IR spectroscopy.
- More information about ellagic acid and other non-metallic crystalline deposits is found here.
Calcium oxalate is another crystalline deposit occasionally found in wine. It occurs when oxalic acid levels in the wine are high enough to precipitate out with the calcium ions present. Oxalic acid levels in wine are usually very low (approximately 70 mg/L), making this a very rarely encountered deposit. Oxalic acid levels can, however, be increased if tartaric acid with oxalic acid impurities is added to the wine, or if corks used contain oxalic acid residues.
Oxalic acid can form stable complexes with iron which prevent its precipitation as the calcium salt. However, with time an increase in the redox potential causes the transformation of the stable ferrous oxalate to the unstable ferric oxalate. The ferric oxalate salt can then release oxalic acid into solution which can combine with calcium and precipitate as calcium oxalate.
Calcium oxalate deposits can be identified using IR spectrometry.
Flavonols are another fairly rare deposit found in wine. They usually appear as yellow-coloured needle shaped crystals in white wines, and principally consist of quercetin with some kaempferol. These phenolic compounds are normally present at low concentrations (37–97 mg/L) in red wines and are not normally present in white wines. They can, however, be extracted into wine from vine leaves, leading to concentrations high enough to cause crystalline instability. The flavonol compounds are initially extracted as soluble glycosides, which hydrolyse slowly under the acidic conditions of wine to give insoluble free flavonols. Because the hydrolysis is slow, flavonol deposits can cause problems by forming after a wine has been bottled. Wines made from grapes that have been mechanically harvested are more susceptible to this type of instablility, as this type of harvesting results in more leaf matter ending up with the grapes.
A test for suspected flavonol deposits is described below. They can also be uniquely identified by IR spectrometry.
|Test for flavonol deposits
Dissolve a portion of the suspected flavonol deposit in methanol and obtain a UV/Vis spectrum from 300 nm to 500 nm of this solution. Add a few crystals of aluminium chloride to the cell and re-run the spectrum. If the absorbance maximum shifts from 370 nm to about 450 nm, the deposit is quercetin.
A small number of cases of flavonol deposits in red wines have been observed at the Institute. In these cases the deposit appeared as a grey/brown sludge which consisted of fine (approximately 1mm thick) needle-shaped crystals. The microscopic appearance of the crystals is shown here. The deposit was insoluble in water, but soluble in ethanol, methanol and sodium hydroxide, the last giving a bright yellow/green colour. The crystals were identified as quercetin dihydrate using X-ray diffraction and IR spectroscopy.
- More information about flavonols and other non-metallic crystalline deposits is found here.
Calcium sulfate does not usually cause problems in table wines, but sometimes causes instabilities in brandies. It precipitates as a white or light-coloured mass, which appears as thin, needle-like crystals under the microscope, and is soluble in hydrochloric acid.
Calcium sulfate forms when calcium is introduced into the brandy often from water containing calcium, which reacts with sulfate anions.
Calcium sulfate deposits can be identified using IR spectrometry.
Calcium mucate is a fairly unusual crystalline deposit, found in wines made from Botrytis-infected grapes. Its appearance is characterised by white clumps, rather than a defined crystal shape.
Calcium mucate deposits form slowly when mucic acid precipitates with calcium present in wine. Mucic acid is a by-product of Botrytis cinerea, thought to be formed by the enzymatic oxidation of galacturonic acid, a major constituent of grape pectin. With heavy rot degradation (10 to 25% of the berries), the pectin can be broken down to produce as much as 1-2 g/L of mucic acid. The acid can form a salt with calcium (calcium mucate) which has very low solubility. It has been suggested that wines containing greater than 0.1 g/L of mucic acid are at risk of calcium mucate precipitation, depending upon the calcium concentration. The calcium content of a table wine usually ranges from 6 to 165 mg/L.
Elimination of Botrytis or other rot-affected fruit is one way to avoid the occurrence of this problem, however this is obviously not possible for winemakers producing Botrytis-style wines. If mucic acid concentration is suspected to be high, calcium carbonate can be added to the wines before bottling, to precipitate out the mucic acid. In addition, reducing sources of calcium in wines is generally a good idea, and will help prevent calcium tartrate as well as calcium mucate problems after bottling.
Calcium mucate deposits can be identified using IR spectroscopy.
Quercetin dihydrate is one of the flavonol compounds which are natural components of grape skins and leaves. Flavonol deposits in wine are relatively rare, but some modern viticultural practices such as increased sun exposure of fruit and machine harvesting can contribute to elevated levels of these compounds in wine (Ziemelis 1982). Quercetin glycosides are extracted from the grape skins during fermentation. The quercetin glycosides then hydrolyse in the acidic wine conditions to release the free quercetin. The quercetin may then crystallise, incorporating some water molecules in the process and form a deposit. The AWRI is not aware of any predictive tests for quercetin deposits.
Ziemelis, G. Flavonol haze-a new form of wine instability arising from technological change. Clarke, J. ed. The Institute of Brewing (Australia and New Zealand section): proceedings of theseventeenth convention; 7-12 March 1982; Perth, WA. Sydney, NSW: The Institute of Brewing (Australia and New Zealand section); 1982: 75-76.