26.2
Fining

26.2.1 Introduction

In the production of wine and other beverages, fining refers to addition of material(s) to remove undesired substances from the beverage. During fining, chemical interactions between fining agent(s) and the targeted component(s) leads to an insoluble product that precipitates from the wine and is removed. Fining agents are usually classified as processing aids rather than additives (Chapter 27) since they are not expected to stay in the wine. In some cases fining is conducted to remove faults in flavor, color, or aroma of the wine, and in other cases the goal is primarily clarification (Table 26.2.1). Fining is rarely entirely selective, and desirable wine components may be partially removed, or “stripped,” from the wine, particularly when excess fining agent is used. Excessive additions of fining agents may also lead to incomplete precipitation of the fining agent (overfining). Overfining can also cause a visible (and undesirable) haze, and can be a health concern for agents with allergenic potential, such that some jurisdictions require labeling when such agents have been used.

Table 26.2.1 Common fining treatments

Winemaking problem	Agent	Issues
Excessive tannin and astringency in red wine	Proteins: gelatin, albumen, isinglass, casein, others	Residual protein, undesired effects on taste and color, allergic potential
Browning or bitterness in white wine	Proteins as above (especially casein and isinglass); polyvinlpolypyrrolidone (PVPP)	Residual protein as above, potentially poor clarification with PVPP
Red color removal for certain white wines (e.g., Pinot Gris, sparkling wines made with Pinot Noir)	Activated carbon	Non‐selective, removes other components
Potential for hazy wine due to heat‐unstable proteins	Bentonite	Metal contamination from bentonite, waste disposal
Off aroma from hydrogen sulfide or volatile thiols	Copper salts (especially sulfate)	Residual copper, reappearance of off aromas, wine oxidation
Off aromas – general	Activated carbon	Non‐selective, loss of other flavors
Poor settling during fining, or visible hazes	Silica gel or kieselsol (co‐fining agents)	Wine pH will affect surface charge

From a chemistry perspective, the key requirements for a useful fining agent are as follows:

• The fining agent must interact with the target compound to form an aggregate. Types of interactions include:
  • Covalent or ionic bonds, as is observed during copper fining of thiols¹
  • Electrostatic bonds, as is observed between bentonite (negative charge at wine pH) and proteins (positive charge)
  • Hydrogen bonding, as can occur during fining of proteins (H‐bond donor) and polyphenols (H‐bond acceptor)
  • Hydrophobic interactions, as occurs between activated carbon and non‐polar wine constituents.
• The fining agent should remove a reasonable portion of the targeted compound, but only in light of the following point.
• The fining agent should display selectivity towards the targeted compound as compared to other organoleptically important compounds. Selectivity may be characterized by a separation factor, or the ratio of the distribution ratios for the targeted and untargeted compounds. In other words, if the fining agent simply absorbs all organic substances, its utility is limited by its poor selectivity.
• The resulting aggregate should have low solubility to ensure precipitation, which is usually accomplished by formation of larger complexes. In some cases, settling aids will be added to neutralize surface charge to allow small aggregates to coalesce, for example, addition of alginates (to neutralize positive surface charge).
• The precipitate should have a density considerably different from wine (usually larger) and a compact structure to favor rapid settling and minimize wine losses. This separation can be facilitated by use of settling aids, for example, SiO₂ (to increase density). Less commonly, flotation using an inert gas like CO₂ can be used to bring the precipitate to the surface to facilitate its removal.

26.2.2 Tannin fining with proteins

During red wine production, the maceration process can result in extraction of excessive tannin or phenolics that contribute to astringency (Chapter 21). The removal of tannin from red wine is generally carried out by addition of protein to yield an insoluble precipitate, decreasing both tannin and perceived astringency. Protein addition may also be used for removing phenolics from white wines, especially in press fractions where they can lead to bitterness or browning.

Several different proteins are employed for removal of tannins and other phenolics, most of animal origin. A common thread of all of these proteins is that they tend to be high in proline and hydroxyproline, which increases their tannin‐binding efficiency as described later in the chapter [1].

• Gelatin is perhaps the most widely used proteinaceous fining agent. Gelatin is produced by partial hydrolysis of collagen, the main structural protein in skin, tendon, and bone. Because of its production method, gelatin does not have a well‐defined chemical structure, but can be classified by its mean molecular weight and gelling strength (bloom number). Typical wine fining applications use gelatin with slightly higher molecular weight (40–50 kDa) and gelling strength (“bloom number” = 100–150) [2] than would typically be used for production of confectionaries.
• Egg whites or albumen purified from the egg whites.
• Casein (or potassium caseinate) sourced from milk. Milk (or milk solids) can also be used, and will possess lipophilicity in addition to tannin binding character; thus, it is often used to co‐remove off‐odorants or other hydrophobic compounds.
• Isinglass, derived from the collagen‐like substance in the swim bladder of certain fish.
• Animal blood (bloodmeal) has been used historically [3], though its use was prohibited after the origin of Creutzfeld‐Jacob (“mad cow”) disease was identified.
• Plant proteins (e.g., from wheat, peas, etc.). Due to the potential for allergies to some of the animal proteins, there is an effort to introduce the use of plant proteins, although some under consideration may also have allergenic issues as well.
• Finally, although technically not a protein, PVPP has structural similarities to proteins and is often used to remove lower molecular weight phenolics.

26.2.2.1 Mechanism of protein–tannin interactions

The mechanism for protein binding to tannin has been proposed to arise from both hydrophobic and polar interactions, and especially hydrogen bonding in the case of proanthocyanidins (Figure 26.2.1). Based on the work of Hagerman and Butler, and McManus and colleagues, Haslam concluded that the binding of tannins to proteins is based on several factors [4]:

• Hydrogen‐bonding appears to be of greater importance for interactions of proanthocyanidins (condensed tannins, Chapter 14) and proteins, with hydrophobic interactions of secondary importance. The reverse appears to be the case for interactions of hydrolyzable tannins (Chapter 13) with proteins [5].
• Binding is stronger when one or both components has a high degree of flexibility, so as to facilitate multidentate binding. For instance, proteins possessing random coil conformations were more effective at binding than tight globular proteins, and flexible polyphenolics, such as proanthocyanidins or pentagalloyl glucose, bind more tightly to proteins than rigid phenolics such as castalagin [6].
• The degree of polyphenol–protein interactions is highly correlated with the proline content of the protein. This appears to be because proline residues provide a site for hydrophobic interactions that initiate binding, which then facilitates hydrogen bonding between protein amide groups and phenolic hydroxyl groups [7].

Figure 26.2.1 Models showing the binding of epigallocatechin gallate to polyproline after 5 minutes (left) and 3 hours (right), as seen via cryo‐TEM images. Reprinted from Food Hydrocolloids, Vol. 20, Poncet‐Legrand et al., Poly(L‐proline) interactions with flavan‐3‐ols units, © 2006, p. 687, with permission from Elsevier [8]

Yokotsuka and Singleton treated different proanthocyanidin fractions with 15 g/hL gelatin, and observed that the highest MW fraction (“polymeric tannin”) was best adsorbed (60% removed) [9], as compared to 48% of oligomeric tannins and only 8% of dimeric proanthocyanidins. The smallest oligomers are thus negligibly affected by fining with proteins [3]. Sarni‐Manchado et al. noted that gelatin was fairly selective in removing larger, gallated proanthocyanidins [10]. Analogously, low molecular weight fractions (2–10 kDa) of gelatin precipitated only 55–70% of the amount of tannin precipitated by standard commercial gelatin (average MW = 70 kDa). Temperatures lower than 25 °C generally resulted in slightly better tannin removal (by ~20%). Typical treatments for gelatin are in the 2–15 g/hL range.

Increasing protein additions results in increased tannin precipitation. However, tannin fining with proteins does not follow Langmuir‐type adsorption behavior, in which adding increasing amounts of protein would result in a limit to what the protein can adsorb. Instead, adsorption of tannins by proteins follows the Freundlich equation (Equation 26.2.1), where addition of tannin results in a decreasing fraction of tannin adsorbed, but no plateau in the amount of tannin bound to the protein (Figure 26.2.2).

(26.2.1)

Figure 26.2.2 Freundlich plot of polymeric tannin adsorption by gelatin (data from Reference [9]). Under Freundlich conditions, a plot of log [solute]/[adsorbent] to log [residual solute] will show a linear correlation with the slope equal to the Freundlich index (1/n) and the intercept the log of the Freundlich constant. See Equation (26.2.1)

In the Freundlich equation, x = total solute concentration (tannin), m is the adsorbent concentration (protein), K is the Freundlich constant (a binding constant value), C is the equilibrium concentration of solute, and n is the Freundlich index for the adsorbent. Values of n less than 1 generally indicate cooperative binding, which is common for protein‐polyphenol interactions.

Key parameters that affect adsorption include the pH of the wine and the pI or isoelectric point (Chapter 5) of the protein. Binding is at a maximum for most protein used in fining when it has a net neutral charge, when the pH is at or near its pI value. However, this is a general rule and the effective pH ranges for binding will depend on protein type [11]. For example, BSA binds only in a narrow pH range (±1.5 pH unit) near its pI, while pepsin, pI = 1.0, is effective from pH 2 to 7, and trypsin, pI = 10.1, has high tannin binding from pH 3 to 10.

The production of white wine often includes fining treatments to remove phenolic components to reduce browning, astringency, or bitterness. One report showed that fining grape juice with protein (K caseinate) decreased polyphenol levels and browning potential when used in conjunction with bentonite and microcrystalline cellulose (as a commercial preparation), compared to bentonite alone [12], with few other effects aside from a slight decrease in protein content and lower concentrations of some volatiles. Casein appears to be much more effective in reducing turbidity, color, and browning potential than gelatin, isinglass, or albumen [13] – possibly because it is more hydrophobic than these other proteins and thus non‐selectively removes other non‐polar compounds. In white wine, none have significant effects on total phenolics or the hydroxycinnamates, the major phenolic fraction in whites (Figure 26.2.3).

Figure 26.2.3 Effect of fining agents on white wine. Percent residual for monomeric, oligomeric, and polymeric proanthocyanidins, total phenol, browning potential, A₄₂₀, removed color in terms of lightness, and turbidity after protein fining treatment. Isoelectric point listed in legend for each protein.

Data from Cosme et al., 2008 [13]

26.2.2.2 Synthetic polymers for phenolic fining

Apart from naturally derived proteins, synthetic polymers with a polyamide structure similar to that of proteins have also been used to remove tannins and other phenolics. Due to its rigidity and porous nature, PVPP demonstrates selectivity towards smaller flavan‐3‐ols, such as monomers and small oligomers, through both hydrogen bonding and non‐polar interactions (Figure 26.2.4). This behavior is in contrast to the preference shown by most proteins towards larger tannins, and PVPP can therefore be used to remove color or decrease browning potential in white wine [14, 15], although its practical application is challenging due to its poor settling characteristics [16]. A typical treatment level is 10 g/hL.

Figure 26.2.4 Hypothetical structure of PVPP, shown as a fragment (six subunits), with hydrophobic and hydrogen‐bonding regions highlighted

26.2.2.3 Co‐fining agents

Protein and related fining agents possess positive surface charges that inhibit aggregation, and also have densities that are only slightly greater than wine (1.2–1.3 g/L versus 1 g/L), resulting in poor settling behavior. This problem can be remedied with co‐fining agents, most commonly silica gel, kieselguhr (a different preparation of silica gel), or polysaccharides such as alginates. Silica gel is often added to wine with or following proteinaceous fining agents, and the two are often combined in proprietary mixtures to improve settling of the fining agent [17]. While silica gel has a negative charge when prepared as kieselsol, the most common type, it appears that at wine pH, the charge is largely neutralized (Figure 26.2.5) [18]. Despite that, it improves flocculation and results in more effective precipitation, probably via hydrogen bonding. When the precipitation occurs, there is an additional clarifying effect due to entrainment of other small particles that are precipitated with the silica–protein complex. Typical treatments are based on commercial suspensions, at rates of 20–100 mL/hL of wine.

Figure 26.2.5 Surface charge of silica gel versus pH. Net charge is near zero at wine pH.

Source: Behren 2001 [18]. Reproduced with permission from AIP Publishing LLC

26.2.2.4 Selectivity

Proteins (and protein‐tannin complexes) can bind to components other than tannin (Table 26.2.1). In general, color is reduced as a result of protein fining treatments, largely because the agents can adsorb pigmented tannin–anthocyanin conjugates (Chapter 24) [19] – greater adsorption is expected from more hydrophobic proteins. On the other hand, protein fining agents do not appear to affect the amount of protein in wine or alter the tendency for that protein to form hazes [20]. However, other reports show that fining with proteins can reduce levels of some aroma compounds by 10–20%, and even more of the glycosides of some important volatiles, potentially diminishing varietal character during aging [21]. Puig‐Diu et al. observed large (25–49%) losses of esters, alcohols, and monoterpenoids when gelatin or bentonite (see below) was used as a fining agent [22]. Sanborn saw a few similar changes in aromatics, but a sensory panel was able to detect only a few differences between control and fined wines in a 26‐attribute descriptive analysis [23].

26.2.2.5 Proteins as allergens

Proteins have allergenic potential, and allergies are known to the various common animal proteins used in fining wine. A trial where standard proteins were added to wines and provided to sensitive subjects elicited very weak responses, possibly because the wine’s acidity denatures the proteins [24]. Regardless, there are now labeling mandates when using such fining agents in winemaking, for instance in Canada [25]. The basis for the labeling is the opportunity for residual proteins to persist when overfining occurs, first reported in 1981 [26]. A recent investigation using a sensitive ELISA assay was unable to detect albumen in a wine that had been normally treated with egg whites [27]. The authors rightly point out that labeling for allergenic content should be based on analytical testing for the presence of allergens. Some investigations have evaluated alternative plant proteins, and while some problems have been noted, such as off‐taste, there are some promising results with pea protein [16] or palatin [28].

26.2.2.6 Inactivated yeast fractions (IVFs) as an alternative to proteins

The inherent association between tannins and other polyphenolics with cell wall mannoproteins [29] is the basis for investigations using IVF, or yeast hulls, as materials to remove phenolics or decrease their astringency in juice or wine. However, the effects observed to date have conflicting outcomes, with explanations that the mannoproteins are either stabilizing or precipitating tannins and pigments [30]. A review of the use of IVF materials demonstrated that the effect is yeast‐strain dependent, and thus the outcome could potentially be difficult to predict, but could be modified by yeast selection [31].

26.2.3 Protein fining with bentonite

One issue with white (or rosé) wine stability is the cloudiness (haze) that can occur when wine is heated (Figure 26.2.6) [32].² This is a result of proteins (Chapter 5) in the wine denaturing and aggregating into larger particles that scatter visible light (the Tyndall effect). This is less of a concern for red wines, since extracted polyphenolic compounds will partially precipitate grape proteins during the winemaking process, and also because the haze is less evident in darkly colored wines. It was discovered early on that only particular proteins contribute to this instability [33, 34]. More recent work points to proteins produced by the grape as a reaction to pathogens [35], particularly thaumatin‐like proteins (TLP) and chitinases. Collectively, these are known as pathogenesis‐related (PR) proteins and can persist through the winemaking process due to their resistance to acid hydrolysis and proteases [36].

Figure 26.2.6 Mechanism for wine haze formation.

Source: Van Sluyter 2015 [32]. Reproduced with permission from American Chemical Society

Aside from the PR proteins, other factors can increase the extent of haze formation.

• The most critical factor appears to be sulfate concentration [37]. Sulfate is a kosmotropic ion (i.e. it interacts strongly with water, making the water less able to solubilize the protein), and high sulfate increases protein denaturation and aggregation.³
• Higher pH will facilitate aggregation, since PR proteins will be positively charged at wine pH (pI > 4).
• High concentrations of phenolics (such as tannins, phenolic acids/esters, and small flavonoids) have also been shown to enhance haze formation [38].⁴

The widely applied remedy to eliminate PR proteins and prevent associated haze problems is to treat wines with bentonite clays, referred to as montmorillonites (the main active component).⁵ Bentonites consist of hydrated aluminum silicate flakes bearing a net negative charge, with the incorporation of exchangeable cations within layers in the crystal structure. Those cations vary according to the source of the bentonite, with some regions predominantly having calcium (e.g., Germany), and others sodium (e.g., Wyoming). The cation content can affect final wine mineral content (Chapter 4), but also affect the performance of the treatment; sodium bentonites swell considerably more when made into a slurry due to the lower charge density of Na⁺ compared to Ca²⁺ in the interlayers (Figure 26.2.7).

Figure 26.2.7 Schematic of the structure of montmorillonite, the mineral in bentonite that provides the adsorptive function. The exchangeable cation, Na⁺, K⁺, etc., is replaced by the protein.

Source: Pusch 2012 [41]. Used under CC‐BY http://creativecommons.org/licenses/by/4.0/

Bentonite can be viewed as a cationic (Na⁺ or Ca²⁺) exchange adsorbent that substitutes its cations for PR proteins, which will be positively charged at wine pH, as described previously. Bentonite contains a quantifiable number of binding sites with a particular binding affinity, and these values can be determined using models developed for enzyme kinetics [42]. Accordingly, protein adsorption most often follows the Langmuir equation with a clear saturation effect (Figure 26.2.8).

(26.2.2)

Figure 26.2.8 Normal Langmuir plots for the bentonite types: sodium (), sodium/calcium (), calcium/sodium (), and calcium () forms.

Source: Blade 1988 [42]. Reproduced with permission of AJEV

The Langmuir equation for adsorption reaches a specific limit. K _L is Langmuir’s constant, C´ is the equilibrium concentration of the solute, x is the total amount of solute, m the concentration of adsorbent, and (x/m)_max the maximum amount that can be absorbed. By plotting C´ versus x/m, one can derive (x/m)_max and K _L. Those values can be obtained from linear regression of a double reciprocal plot [42].

The different cationic forms of bentonite lead to differences in protein binding affinity. Beyond having a greater swelling ability, sodium bentonites can also adsorb a larger amount of protein for a given mass (e.g., about twice as much as bentonites containing calcium, Figure 26.2.8). On the other hand, calcium bentonites give more compact lees due to their lesser swelling. Typical treatment levels are 20–50 g/hL in white wines.

Sodium or calcium will be released during bentonite fining, as is expected based on the ion‐exchange mechanism, which could potentially cause legal and flavor issues (sodium) or stability issues (calcium). Bentonites contain between 3 and 12% sodium and 3 and 17% calcium, depending on whether the sample is a calcium or a sodium type. The amount of sodium that appears in a wine as the result of treatment is reportedly 15–25 mg/L and 20–30 mg/L for calcium, when the wine is treated with high levels of 400 g/hL of sodium‐containing bentonites [43], although others have used exceptionally high levels of treatment for experimental purposes, 2500 g/hL, and observed an increase of 300–400 mg/L of sodium [44]. Beyond that, these reports also show that other contaminant metals also appear (Chapter 4), such as lead, aluminum, or iron.

26.2.3.1 Selectivity

In addition to removing proteins, bentonite can strip other wine components in a non‐selective fashion, but a review suggests only marginal effects that wine consumers might be able to discern when used at normal levels [35].

26.2.3.2 Alternatives to bentonite

Bentonite disposal is a problem for wineries and various alternatives for protein removal (including more effective uses of bentonite) have been proposed, but none have been adopted to any significant extent. A report reviewed potential substitutes for bentonite [32]. Proteases were considered promising when used with flash pasteurization, as there were few sensory changes and the resulting wines did not turn hazy. Carrageenan has some potential but it has some hazing potential itself, while chitin has several drawbacks. Zirconium oxide could function as substitute ion exchange material for bentonite, and it could be reused as well, but it must be tested in a commercial setting to establish viability.

26.2.4 Miscellaneous fining and related treatments

26.2.4.1 Copper fining and sulfurous malodors

Copper salts are used to treat some sulfur aromas in wine that have potent aroma effects. In particular, low molecular weight sulfur compounds including H₂S and thiols are particularly noxious (Chapters 10 and 30). H₂S will react with copper salts (such as CuSO₄) to form an exceptionally insoluble copper sulfide (CuS, K _sp = 4 × 10⁻³⁶). In contrast to H₂S and thiols, copper will not form a complex with sulfides, disulfides, or thioacetates. Although CuS and related complexes are highly insoluble, they appear to remain dispersed in wine. Commercial white wines adjusted to ~1 mg/L Cu and equimolar H₂S showed essentially unchanged Cu content (>95% of original value) following filtering or 5 days storage and racking [45] (Chapter 24). Furthermore, copper complexes appear to be capable of regenerating H₂S and possibly thiols during storage [46]. Thus, copper additions are not necessarily a “fining” operation, as the copper remains, albeit in a different form.

26.2.4.2 Removal of transition metals

Historically, a number of methods have been used to remove iron and copper in order to avoid the formation of precipitates (casses) [47]. However, the universal usage of stainless steel and wood cooperage in wine production today has essentially eliminated the need for such treatments. During the middle parts of the twentieth century, the use of epoxy‐coated steel tanks or concrete tanks with exposed steel reinforcement led to dissolved iron at levels of 5–20 mg/L [48]. At those levels iron casse (iron (III) phosphate [white casse] or tannate [blue casse]) could readily form in bottled wine, but with iron levels in modern times at 0.5–3 mg/L (Chapter 4), winemakers have largely forgotten about the problem or its treatment. The most effective treatment involves fining with potassium ferrocyanide, which forms an insoluble complex with metals such as iron and copper [47]. The treatment is complicated because the reagent has the potential to release cyanide if not used properly (i.e., if overfined), so its permitted use is tightly regulated. Ion exchange resins also have some utility in removing metals, which was discussed in Chapter 26.1.

26.2.4.3 Activated charcoal and non‐polar sorbents

When a wine becomes contaminated with various undesirable components, such as aroma defects from rotten grapes (Chapter 18), a winemaker’s last resort is often activated carbon or charcoal. Activated carbon is a non‐specific adsorbent that has a very high surface area and can adsorb non‐polar and polar substances. It will remove many components in addition to the problematic substance, so it is generally only used in small quantities or applied when a neutral tasting product is better than the untreated option [49]. Similar effects can be achieved through the use of non‐polar resins or neutral oils (milk or cream), although these are not approved for use in winemaking in some regions (Chapter 27).

Although various taints can be removed by activated carbon or non‐polar materials, the efficacy of these treatments is compromised by their poor selectivity. For example, 3‐isopropyl‐2‐methoxypyrazine (IPMP, “MALB taint,” Chapter 5) can be decreased by >30% with 0.2 g/L activated charcoal [49]. However, sensory testing revealed no change in the MALB taint character, likely because other desirable odorants (e.g., esters) with similar or greater hydrophobicity were also removed. For juice taints, these problems can be avoided by treating the juice with a non‐polar sorbent rather than the wine, since most wine odorants are formed during fermentation (see the Introduction in this chapter) [50].

Hydrophobic materials are particularly well suited for removing highly hydrophobic compounds, including several undesirable components in wine. For example, TCA (“cork taint,” Chapter 18) has a log P ~4.0, and is thus one of the most non‐polar compounds in wine – by comparison, the log P of ethyl hexanoate is estimated to be 2.8. Both cork particles and polyethylene have been reported to be fairly effective at diminishing levels of TCA and several related compounds [51], and a number of companies supply filtration materials with hydrophobic resins designed to remove cork taint [52]. The highly non‐polar TDN (log P ~ 4.8, “petrol,” Chapter 9) is reported to decrease 50% due to scalping by synthetic closures, while several less polar aroma compounds were negligibly affected [53]. Analogously, activated carbon has been applied to the removal of ochratoxin A (Chapter 18) [54]. In all of these cases, relatively small rates of hydrophobic sorbent are necessary to remove sufficient quantities of the targeted highly hydrophobic compounds, which minimizes the effects on non‐targeted compounds.

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Notes

1 For simplicity, many practical wine texts imply that all interactions are electrostatic, that is, all fining agents and target compounds are either positively or negatively charged. While appropriate for an introductory level, this approximation is not useful in describing more complex behavior.

2 This is usually inadvertent, such as from incorrect storage conditions or during transit. Formation of protein haze is primarily a visual nuisance as most consumers expect wines to be clear, irrespective of whether the wine may be abused once it leaves the retailer (e.g., placed in the trunk (boot) of a car on a hot day).

3 For similar reasons, ammonium sulfate gradients are widely used in biochemistry to induce precipitation (“salt out”) different fractions of proteins.

4 Complexes of polyphenols (particularly flavan-3-ol dimers) with proteins appears to be the major cause of beer haze [39].

5 The use of related aluminosilicate clays (kaolin, Spanish clay) is frequently described in nineteenth century winemaking texts, although the application was to clarify hazy wines rather than in its current role as a prophylactic treatment. The use of bentonite for clarifying wine was first described in the literature by Saywell in 1934 [40], and it has largely displaced the use of other clays due to its higher adsorptive capacity.