Ash Analysis

16
G. Keith Harris

Contents
16.1    Introduction 261
16.1.1 Definitions 262
16.1.2 Importance of Ash in Food Analysis 262
16.1.3 Ash Contents in Foods 262
16.2    Methods 262
16.2.1 Sample Preparation 263
16.2.2 Dry Ashing 263
16.2.3 Wet Ashing 265
16.2.4 Microwave Ashing 265
16.2.5 Other Ash Measurements 267
16.3    Comparison of Methods 267
16.4    Summary 268
16.5    Study Questions 268
16.6    Practice Problems 269
References 270

16.1 INTRODUCTION and lost during dry ashing. Wet ashing methods often involve
the use of pressure vessels to contain acids and samples,
Ashing is an important step in proximate or specific mineral allowing for higher temperatures and faster processing.
analysis. Ash refers to the inorganic (mineral) residue remain- Microwave systems also can be used for both dry and wet
ing after complete combustion or complete acid-­facilitated ashing. In either case, microwave systems tend to speed up
oxidation of organic matter in food. A basic knowledge of the the ashing process, although sample throughput may be a lim-
characteristics of ashing procedures and types of equipment iting factor. Most dry samples (i.e., whole grain, cereals, and
is essential to ensure reliable results. Two major types of ash- dried vegetables) need no preparation for ashing, while fresh
ing are used: dry ashing and wet ashing. Dry ashing is used as vegetables and other high-moisture foods are generally dried
part of proximate analysis to estimate total mineral content prior to ashing to prevent rapid boiling and sample loss. High-
and as a preparatory step for specific mineral analyses. Dry fat products, such as meats, may need to be dried and fat-­
ashing requires very high (500–600 °C) temperatures, which extracted before ashing to prevent smoke generation during
may be achieved by conventional or microwave heating. Wet heating steps. Flammable volatiles, like ethanol, should also
ashing combines acid-facilitated oxidation and lower tem- be eliminated prior to ashing. Ashing is a gravimetric analy-
peratures (100–120 °C) to eliminate organic material and is sis, in which the final (ashed) weight is compared to the origi-
required for the analysis of minerals that might be volatilized nal weight of the sample. The ash content of foods can then
be expressed on either a wet weight or a dry weight basis. For
G. K. Harris (*) general and food-specific information on measuring ash con-
Department of Food, Bioprocessing and Nutrition Sciences, North tent for humans and animals, see references [1–16].
Carolina State University, Raleigh, NC, USA
e-mail: [email protected]

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2024 261
B. P. Ismail, S. S. Nielsen (eds.), Nielsen’s Food Analysis, Food Science Text Series,
https://doi.org/10.1007/978-3-031-50643-7_16
262 G. K. Harris

16.1.1 Definitions Table 16.1 Ash content of selected foods

Percent ash (wet weight
Dry ashing refers to the use of a muffle furnace capable of Food item basis)
maintaining temperatures of 500–600 °C. Water and vola- Cereals, bread, and pasta
tiles are vaporized, and organic substances are burned in the  Rice, brown, long grain, raw 1.5
 Corn meal, whole grain, yellow 1.1
presence of oxygen to form CO2 and oxides of N2. In this  Hominy, canned, white 0.9
way, organic materials are removed from the sample. The  White rice, long-grain, regular, raw, 0.6
remaining minerals are converted to oxides, sulfates, phos- enriched 1.6
phates, chlorides, and silicates. Some elements partially vol-  Wheat flour, whole grain 0.9
 Macaroni, dry, enriched 2.5
atilize with this procedure, so other methods must be used if  Rye bread
ashing is a preliminary step for the analysis of these Dairy products
minerals.  Milk, reduced fat, fluid, 2% 0.7
Wet ashing is a procedure used to chemically oxidize and  Evaporated milk, canned, with added 1.6
remove organic substances using strong acids, oxidizing vitamin A 2.1
 Butter, with salt 0.7
agents, and lower temperatures (100–120 °C) than for dry  Cream, fluid, half and half 2.0
ashing. The acids and oxidizing agents used must also be  Margarine, hard, regular, soybean 1.1
capable of solubilizing the minerals to be analyzed.  Yogurt, plain, low fat
Hydrochloric, sulfuric, and nitricacids are commonly used. Fruits and vegetables
Perchloric acid was commonly used in the past for wet ash-  Apples, raw, with skin 0.2
 Bananas, raw 0.8
ing (AOAC Method 975.03), but safety concerns over its for-  Cherries, sweet, raw 0.5
mation of explosive peroxides have largely eliminated its use  Raisins 1.9
(so it will not be further discussed in this chapter). Since wet  Potatoes, raw, skin 1.6
ashing is conducted at lower temperatures than dry ashing  Tomatoes, red, ripe, raw 0.5
Meat, poultry, and fish
and may use sealed pressure vessels, minerals are not lost
 Eggs, whole, raw, fresh 0.9
due to volatility. For this reason, wet ashing is preferable to  Fish fillet, battered or breaded and 2.5
dry ashing as a preparation for comprehensive mineral anal- fried 0.9
ysis of samples.  Pork, fresh, leg (ham), whole, raw 1.9
 Hamburger, regular, single patty, plain 1.0
 Chicken, broilers or fryers, breast meat 1.1
only, raw 6.8
16.1.2 Importance of Ash in Food Analysis  Beef, chuck, arm pot roast, raw
 Beef jerky
Ash represents an estimate of the total mineral content of From the US Department of Agriculture, Agricultural Research Service
foods. Determining ash content is important for several [16], Food Data Central fdc.nal.usda.gov
reasons. First, it is used to determine the total mineral con-
tent of foods as part of proximate analysis. Second, ashing
16.1.3 Ash Contents in Foods
is used to prepare food samples for specific mineral analy-
sis, whether for essential nutrients or for toxic heavy met-
The ash content of food can range from 0 to 12%. The high-
als. Because certain foods are high in particular minerals,
est ash values are found in dry foods, such as beef jerky or
ash analysis can be important from nutritional, food qual-
dried vegetables, but rarely exceed 5% for fresh, moisture-­
ity, and toxicological standpoints. For example, dairy and
containing foods. The average ash content for various food
beef are known to be rich sources of calcium and iron,
groups is given in Table 16.1.
respectively. The mineral content of plant foods tends to be
more variable than that of animal products and is depen-
dent on the soil in which it is grown. For example, rice
16.2 METHODS
grown in arsenic-containing soil effectively concentrates
it. High levels of transition metals in lipid-rich foods may
Principles, materials, instrumentation, general procedures,
speed rancidity and limit shelf life. While ash is not
and applications are described below for various ash deter-
reported directly on foods for human consumption, it is
mination methods. Refer to the methods cited for detailed
commonly part of the ingredient specifications for flours
descriptions of the procedures.
and wholegrains and may be reported on pet food labels.
16 Ash Analysis 263

16.2.1 Sample Preparation form a crust on heating, one or two drops of refined olive oil
(which contains no ash) may be added to allow steam to
Ash analysis does not require large sample sizes. A 2–10 g escape as the crust forms.
of sample is generally sufficient. Some newer instruments Smoking and burning may occur when products high in
allow for sample sizes as low as 250 mg. For this reason, it fat and protein (e.g., cheese, spices, and peanuts) are dry-­
is necessary to acquire a homogenous, representative sam- ashed. This process is allowed to finish slowly by keeping
ple for accurate results. Milling, grinding, and other meth- the muffle door slightly open until combustion stops, then
ods used to reduce particle size and homogenize samples the door is fully closed, and the normal ashing protocol is
will not significantly alter ash values. In contrast, mineral proceeded. High-fat samples may also be ashed after drying
contamination from the environment or from grinding and fat extraction to minimize smoke production. In most
equipment can introduce error, but the use of sample cases, mineral loss is minimal during drying and fat extrac-
blanks can account for this. (This is particularly true of tion. Fat-extracted samples should not be heated until flam-
metals such as iron, since most grinders and mincers are of mable extraction solvents (e.g., hexane and ether) have been
steel construction.) Crucibles and glassware also can be a evaporated completely to avoid the potential risk of solvent
source of contaminants unless they are carefully cleaned ignition or explosion. All of these procedures require the use
between samples. This can be addressed by soaking cru- of fume hoods to avoid release of smoke into the laboratory.
cibles and glassware in an acid bath to solubilize mineral Most of these macronutrient-related issues apply to dry ash-
contaminants and rinsing repeatedly with distilled-deion- ing, which is described in more detail in the section that
ized water. The crucibles themselves can also be “pre- follows.
ashed” in a muffle furnace to remove organic
contaminants.
16.2.2 Dry Ashing
16.2.1.1 Plant Materials
Plant materials are generally oven-dried prior to grinding 16.2.2.1 Principles and Instrumentation
and ashing to remove moisture. Since dried samples often Dry ashing is incineration at high temperature (525 °C or
are used for multiple determinations (e.g., protein, fiber, and higher). Sample incineration is accomplished with a muf-
lipid), it may be necessary to keep oven temperatures at or fle furnace, an oven that can reach temperatures in excess
below 100 °C to prevent the destruction or alteration of non- of 1000 °C. Several models of muffle furnaces are avail-
mineral analytes. Fresh stem and leaf tissue should be dried able, ranging from large capacity units requiring either
in two stages (i.e., first at a lower temperature of 55 °C and 208- or 240-V electric supplies to small benchtop units
then at a higher temperature) to prevent formation of artifact utilizing 110-V outlets. The muffle furnace must be placed
lignin and other undesired products. Plant material with 15% in a fume hood and may have to be placed in a heat-proof
or less moisture may be ashed without prior drying. Low-­ room along with drying ovens. It is important that large
temperature drying can be accomplished via the use of a muffle furnaces are equipped with a double-pole, single-
muffle furnace equipped with temperature gradient settings. throw switch. Heating coils are generally exposed, and
Samples can thus be dried at low temperature and then ashed care must be taken when taking samples in and out with
in the same crucible. metal tongs.
Crucibles are used to contain dry ashing samples. Lids are
16.2.1.2 Macronutrient Composition Effects sometimes used to better contain samples. Crucible selection
Animal products, syrups, and spices require special consid- is critical in ashing because the type depends on the specific
eration prior to ashing because of high fat, protein, moisture, use. Primary considerations relate to chemical stability and
or high sugar content that may result in loss of sample and resistance to high temperatures. Quartz crucibles are resis-
underestimation of ash content due to spattering, swelling, or tant to acids and halogens, but not alkali, at high tempera-
foaming. Conversely, high-sugar foods may form intractable tures. Vycor® brand crucibles are stable to 900 °C, but
tars that can lead to overestimation of ash. Depending on the Pyrex® Gooch crucibles are limited to 500 °C. Ashing at a
application, crucible covers or the use of sealed crucibles lower temperature of 500–525 °C may result in slightly
may be used to contain samples. Meats, sugars, and syrups higher ash values due to incomplete decomposition of car-
should be evaporated to dryness on a steam bath or with an bonates. Porcelain crucibles resemble quartz crucibles in
infrared (IR) lamp prior to ashing. For products that tend to their properties but will crack with rapid temperature
264 G. K. Harris

changes. Since they are relatively inexpensive, porcelain cru- gradually by sliding them to one side to prevent a sudden inrush
cibles are often the crucible of choice. Steel crucibles are of air. Covers with a ground-glass sleeve or fitted for a rubber
resistant to both acids and alkalis and are inexpensive, but stopper allow for a slower release of a vacuum.
they are composed of iron, chromium, and nickel, which can The ash content is calculated as follows:
erroneously increase apparent ash content. Platinum cruci-
bles are very inert and are probably the best overall, but they ( WAA − TWOC )
%Ash ( dry basis ) = ×100 (16.1)
are currently far too expensive for routine use for large num- ( OSW x DMC )
bers of samples. Quartz fiber crucibles are disposable and
unbreakable and can withstand temperatures of up to where:
1000 °C. They are porous, allowing air to circulate around
the sample and speed combustion. This reduces ashing times WAA = weight after ashing
significantly and makes them ideal for solids and viscous liq- TWOC = tare weight of crucible
uids. Quartz fiber also cools in seconds, virtually eliminating OSW = original sample weight
the risk of burns. DMC =
dry matter coefficient (percent sample
All crucibles should be marked for identification. Marks solids/100)
on crucibles with a felt-tip marking pen will disappear dur-
ing ashing in a muffle furnace. Laboratory inks scribed with Consider one of three options for ash calculations. Use
a steel pin are available commercially. Crucibles also may be the DMC, or dry matter coefficient, to directly convert wet
etched with a diamond point and marked with a 0.5 M solu- weight percent ash values to dry weight ash values. For
tion of FeCl3, in 20% HCl. An iron nail dissolved in concen- example, if corn meal is known to be 87% dry matter, the
trated HCl forms a thick brown pigment that is a satisfactory dry matter coefficient would be 0.87. Dry weight ash con-
marker. The crucibles should be fired and cleaned prior to tent can be determined without the DMC if samples are
use. dried at low temperatures in the same crucible prior to ash-
ing. The denominator becomes (dry sample weight – tared
16.2.2.2 Procedures crucible weight) and values are reported as dry weight.
AOAC International has several dry ashing procedures (e.g., This method will be more accurate than the DMC method
AOAC Methods 900.02 A or B, 920.117, 923.03, 942.05) for for dry weight determination but adds an extra, time-con-
certain individual human and animal foodstuffs. The general suming step. If DMC values are unknown and samples
procedure includes the following steps: have not been predried, the dry matter coefficient is deleted
from the denominator and percent ash values are reported
1. Weigh a 5- to 10-g sample into a tared crucible. Account on an as-­ received, fresh, or wet weight basis, as
for the weight of crucible covers if they are used. Predry appropriate.
high-moisture samples.
2. Place crucibles in cool muffle furnace using tongs. Use 16.2.2.3 Special Applications
tongs, gloves, and protective eyewear if the muffle fur- Some AOAC procedures recommend steps in addition to
nace is warm. those listed previously. If carbon is still present following the
3. Heat samples for 12–18 h (or overnight) at about 550 °C. initial ashing (residual carbon will appear as a black, tar-like
4. Turn off the muffle furnace. Wait until the temperature material), several drops of water or nitric acid should be
has dropped to 250 °C or lower before opening the fur- added and samples should be re-ashed. If the carbon persists,
nace door. Open the door slowly to avoid the loss of the which may occur with high-sugar samples, follow this
powdery ash that may result from rapid air movement. procedure:
5. Using tongs, quickly but carefully transfer crucibles to a
desiccator with a porcelain plate and desiccant. Cover 1. Suspend carbon-containing ash samples in water.
crucibles, close desiccator, and allow crucibles to cool to 2. Filter samples through ashless filter paper because this
room temperature prior to weighing. residue tends to form a glaze.
3. Dry the filter and filtrate together.
Note 4. Place paper and dried filtrate in muffle furnace and re-­
Avoid placing hot samples into desiccators for two reasons. ash, subtracting the weight of the filter paper to determine
First, this could cause sudden displacement of the desiccator ash percent.
cover if sufficient pressure is generated. Second, as hot samples
cool, a vacuum may form, making it difficult to remove the Other suggestions that may be helpful to accelerate the
desiccator cover. Desiccator covers should always be removed ashing process:
16 Ash Analysis 265

1. High-fat samples should be solvent-extracted either using 2. Prepare blanks (3 mL H2SO4 plus 5 mL HNO3) to be run
a crude fat determination procedure or, less ideally, by with all samples sets.
burning off lipids prior to closing the muffle furnace. 3. Add 3 mLH2SO4 followed by 5 mL HNO3 to sample
2. Mix high sugar, high-moisture samples, like jelly, with flasks.
cotton wool to avoid spatter. 4. Heat samples to boiling on a hot plate. Brown-yellow
3. Salt-rich foods may require a separate ashing of water-­ fumes will be observed.
insoluble components and salt-rich water extract. Use 5. Once brown-yellow fumes cease and white fumes from
crucible covers to prevent spattering. decomposing H2SO4 are observed, samples will darken.
4. An alcoholic solution of magnesium acetate can be added Remove flasks from the hot plate but do not allow them to
to accelerate ashing of cereals. An appropriate blank cool to room temperature.
determination is necessary. 6. Slowly add 3–5 mLHNO3.
7. Put flasks back on hot plates and allow HNO3 to boil off.
The advantages of dry ashing are that it is a relatively safe Proceed to the next step when all HNO3 has been removed
and inexpensive method, that it requires no acids, added (samples will be clear or straw yellow in color). If the
reagents or blank subtraction, that sample recovery is high, solutions remain dark in color, add another 3–5 mL HNO3
and that little attention is needed once ignition begins [2]. to flasks and reboil. Repeat the process until the clear to
Depending on the oven size, a large number of crucibles can straw yellow color is obtained.
be handled at once, and the resultant ash can be used addi- 8. Continue boiling to reduce the volume appropriately to
tionally in other analyses for most individual elements, acid-­ allow for ease of final transfer. Allow samples to cool to
insoluble ash, and water-soluble and insoluble ash. The room temperature, then quantitatively transfer samples to
disadvantages of dry ashing are the length of time required appropriately sized volumetric flasks.
(12–18 h or overnight) and the expense of equipment relative 9. Dilute samples to volume with ultrapure water and mix
to traditional wet ashing. There will be some loss of select well. Dilute further, as appropriate, for the specific min-
elements due to volatilization and to interactions between eral analyses.
mineral components and crucibles. Volatile elements at risk
of being lost include As, B, Cd, Cr, Cu, Fe, Pb, Hg, Ni, P, V, A combination dry and wet ash procedure is given in
and Zn. AOAC Method 985.35 “Minerals in Infant Formula, Enteral
Products, and Pet Foods.”
There are at least two advantages to using the wet ashing
16.2.3 Wet Ashing procedure over dry ashing: (1) minerals typically stay in
solution throughout the procedure, making them easier to
16.2.3.1 Principles, Materials, and Applications transfer for specific mineral analyses, and (2) wet ashing is
Wet ashing is sometimes called wet oxidation or wet diges- faster than dry ashing. The disadvantages of wet ashing, by
tion. Its primary uses are to get a more accurate, comprehen- the open vessel hot plate method, over dry ashing are that:
sive representation of percent ash than from dry ashing and (1) this type of wet ashing requires virtually constant opera-
to prepare for specific mineral analyses post-ashing. tor attention, (2) corrosive reagents are used, requiring addi-
The phasing out of perchloric acid for wet ashing (men- tional safety precautions, caustic waste disposal, and personal
tioned in Sect. 16.1.1) as a sole oxidizing solvent has led to protective equipment, and (3) only small numbers of samples
the use of other acids. Unfortunately, using a single acid for can be handled at any one time.
wet ashing does not give complete and rapid oxidation of
organic material. So, different concentrations and ratios of
nitric and sulfuric acids, sometimes with hydrogen peroxide 16.2.4 Microwave Ashing
added, are often employed.
Both dry and wet ashing can be done using microwave heat-
16.2.3.2 Procedures ing, instead of the conventional dry and wet ashing proce-
This is a wet ash procedure using concentrated nitric and dures described previously. Companies like the CEM
sulfuric acids (must be performed in a fume hood) (John Corporation (Matthews, NC) and Anton-Paar (Graz, Austria)
Budin, Silliker Laboratories, Chicago, IL, personal have developed a series of microwave-heated instruments for
communication): accelerated dry and wet ashing procedures. While ashing by
conventional means can take many hours, the use of micro-
1. Accurately weigh a dried, ground 1 g sample in a 125-mL wave heating can reduce sample preparation time to minutes,
Erlenmeyer flask (previously acid-washed and dried). allowing laboratories to increase their sample throughput
266 G. K. Harris

significantly (current models have a throughput of 5–24 sam- ditions in real time. This closed-vessel microwave ashing
ples per hour). This advantage has led to widespread use of approach was used to improve AOAC Method 984.27, which
microwave ashing, especially for wet ashing, within both had previously used conventional wet or dry ashing to pre-
analytical and quality control laboratories in food pare samples for ICP analyses [14].
companies. Typically, in a closed-vessel microwave system, samples
are placed in vessels with the appropriate amount of acid.
16.2.4.1 Microwave Wet Ashing Vessels are sealed and loaded into the carousel that is then
Microwave wet ashing (acid digestion) may be performed placed in the microwave cavity. Floor-mounted sensors are
safely in either an open- or closed-vessel microwave system. used to measure the temperature of each vessel as it passes
The choice of open vs closed systems depends on the amount over the sensor. Adjustable settings may include time, tem-
of sample and the temperatures required for digesting. Due perature, and power in multiple stages. Digestions normally
to the ability of the closed vessels to maintain high pressures take less than 30 min. Because of the pressure generated by
(up to 1500 psi), acids may be heated well past their boiling raising the temperature of a reaction, vessels must be allowed
points. The samples generate the pressure based on decom- to cool before being opened. The ability to process multiple
posing in the acid and producing CO2 and NOx gases. The samples simultaneously provides the chemist with greater
vessel then holds this pressure, which allows for tempera- throughput than traditional methods. (Note that some closed-­
tures above the boiling point. This ensures fast, complete dis- vessel microwave digestion systems may also be used for
solution of hard-to-digest substances. It also allows the solvent extraction, protein hydrolysis, and chemical synthe-
chemist to use nitric acid with samples that might normally sis with the proper accessories.)
require harsher acid mixtures, such as sulfuric or perchloric
acids. Closed vessels specifically designed for high-­ 16.2.4.2 Microwave Dry Ashing
temperatures/high-pressure reactions, nitric acid can reach a Compared to conventional dry ashing in a muffle furnace,
temperature of 240 °C (However, the methods specified by which often takes many hours, microwave muffle furnaces
the equipment manufacturers, FDA, and AOAC use 200° C.). (Fig. 16.2) can ash samples in minutes, decreasing analysis
Nitric acid is often the acid of choice for microwave diges- time by as much as 97%. Microwave muffle furnaces can
tion, though hydrochloric, hydrofluoric, and sulfuric acids reach temperatures of up to 1200 °C. These systems may be
are also used, depending on the sample and the subsequent programmed with various heating and cooling methods. In
analysis being performed. Closed-vessel microwave diges- addition, they are equipped with exhaust systems that circu-
tion systems (Fig. 16.1) can process up to 40 samples at a late the air in the cavity to help decrease ashing times. Some
time, with vessel liners available in Teflon®, TFM™ fluo- also have scrubber systems to neutralize harmful fumes, thus
ropolymer, and quartz. Closed-vessel wet ashing systems eliminating the need for and space taken up by fume hoods.
allow for control of time, temperature, and pressure param- Any crucible that may be used in a conventional muffle fur-
eters. Some instruments enable the user to adjust these con- nace may be used in a microwave furnace, including those

Fig. 16.1 Microwave closed-vessel digestion system. (MARS6; Fig. 16.2 Microwave muffle furnace. (Phoenix BLACK; Courtesy of
Courtesy of CEM Corporation, Matthews, NC) CEM Corporation, Matthews, NC)
16 Ash Analysis 267

made of porcelain, platinum, quartz, and quartz fiber. Some 2. Ash Insoluble in Acid. After dry-ashing a sample, the
systems can process up to 15 (25-mL) crucibles at a time. ash is solubilized in 10% HCl, boiled, filtered, and the
Typically, in microwave dry ashing, a pre-ashed, dried insoluble material is re-ashed. This may be used as a mea-
crucible (one that has been stored in a desiccator) is weighed, sure of the surface contamination of fruit, vegetables,
sample is added, and the crucible is weighed again. The cru- wheat, and rice coatings. Contaminants are generally sili-
cible and sample are placed in the microwave furnace, and cates and remain insoluble in acids, except HBr.
time and temperature parameters are set. As with microwave-­ 3. Alkalinity of Ash (e.g., AOAC Method 900.02, 940.26).
heated wet ashing, settings may include power, temperature, Dry-ashed samples are treated with a known amount of
and pressure gradients. The system is started, and the pro- 0.1 N HCl, then boiling water is added. Samples are
gram is run to completion. The crucible is carefully removed titrated with 0.1 N NaOH using a methyl orange indica-
with tongs and reweighed. Tongs serve a dual purpose. They tor. The volume of NaOH required indicates the alkalin-
prevent contact with hot crucibles and prevent residues from ity of the ash. The ash of fruits and vegetable is alkaline,
the technician’s hand or gloves from contaminating samples. while the ash of meats and some cereals is generally
The sample then may be further analyzed, if necessary. Some acidic.
tests call for acid to be added to a dry-ashed sample, which 4. Sulfated Ash (AOAC Method 900.02, 950.77). This
may serve to enhance digestion or to solubilize minerals for method, applied mostly to sugars, syrups, and color
further analysis. additives, measures the amount of residual substance
A comparative study [15] showed that dry-ashing various not volatilized when the sample is ignited in the pres-
plants for 40 min using a microwave system (CEM ence of sulfuric acid. Samples are wetted with sulfuric
Corporation, Matthews NC) was similar to the 4 h time in a acid and heated on a hot plate and then in a muffle fur-
conventional muffle furnace. A time of 20 min was shown to nace. This procedure is repeated until a constant weight
be adequate for the plant material used except for Cu deter- is achieved, with the final weight expressed as % sul-
minations, which required the full 40 min to obtain similar fated ash. (The procedure also can be performed in a
results. Other comparative examples include dried egg yolks, microwave muffle furnace, e.g., CEM Corporation,
which can be ashed in 20 min in a microwave system but Fig. 16.2).
require 4 h in a conventional muffle furnace. It takes 16 h to
ash lactose in a conventional muffle furnace, but only 35 min
in a microwave furnace. Though microwave furnaces may 16.3 COMPARISON OF METHODS
not hold as many samples as a conventional furnace, their
speed actually allows significantly more samples to be pro- The four major ashing methods described in this chapter
cessed in the same amount of time. One additional advantage (dry and wet ashing using conventional vs microwave heat-
is that microwave furnaces typically do not require fume ing) are summarized and compared in Table 16.2. Dry ash-
hood space. ing requires a muffle furnace, is more time-consuming, and
uses more electricity but is less hazardous than wet ashing
on a hot plate. Wet ashing using a hot plate requires a fume
16.2.5 Other Ash Measurements hood, use and disposal of corrosive reagents, and constant
operator attention. While wet oxidation causes little volatil-
The following are several special ash measurements and ization of minerals, dry ashing will result in the loss of
their applications: volatile elements. Both dry and wet ashing can be done
using microwave systems that require relatively expensive
1. Soluble and Insoluble Ash (e.g., AOAC Method 900.02). instrumentation but greatly reduce the time needed for ash-
After dry-ashing a sample, ash is solubilized in boiling ing and generally do not require use of a fume hood. If the
water and filtered. Water-soluble and water-insoluble intent is further elemental analyses, the specific elements
fractions are then re-ashed. Soluble and insoluble ash are being analyzed will dictate whether wet or dry ashing is
indices of the fruit content of preserves and jellies. Lower used. Some minerals micronutrients and most volatile ele-
water-soluble ash indicates a higher percentage of fruit, ments will require special equipment and procedures. Refer
since fruit contains a variety of minerals that could add to to Chaps. 9 and 21 for specific preparation procedures for
the insoluble fraction. mineral analyses.
268 G. K. Harris

Table 16.2 Summary of common ashing methods

Ashing
method Principle Sources of error Advantages Disadvantages Applications
Dry ashing Sample heated to very Microelement Can analyze many Slow (takes 12–18 h). Total ash content for
high (500–600 °C) contamination (from samples at once. Some minerals proximate
temperature. All grinder or water used to Requires little volatilized. Minerals analysis. May be
organic matter clean crucibles). Sample technician time. are difficult to used as
incinerated. loss during pre-ash Safe. No blanks resolubilize preparation for
Remaining inorganic drying step. Volatilization needed specific mineral
material quantitated of some elements. analysis
gravimetrically Incomplete combustion
Wet ashing Organic matter oxidized Microelement Shorter time (~2 h) Requires constant Ashing of samples
using acids and contamination. Must run than dry ashing. attention. Lower prior to mineral
oxidizing agents, blanks to correct for Minerals stay in sample throughput analysis for
leaving inorganic organic matter in acid solution. Little or than dry ashing. Can official analyses
matter and oxidizing agent. no volatilization be dangerous. Use of
Sample loss may occur strong acids and
due to spattering oxidizers. Sample
loss due to spattering
Microwave Microwave energy heats Microelement More rapid Costly. Lower sample Determine total ash
ashing sample to very high contamination (from (~30 min) than throughput than content for the
(dry) temperatures, grinder or water used to regular dry regular dry ashing. determination of
incinerates organic clean crucibles). Sample ashing Some minerals proximate
matter, leaves loss during pre-ash volatilized. May be composition or for
inorganic matter to be drying step. Volatilization hard to resolubilize quality control
quantitated of elements. Incomplete purposes
gravimetrically combustion
Microwave Microwave energy and Microelement Takes less time Expensive. Can handle Rapid ashing of
ashing acid (and sometimes contamination. Must run (~30 min) than fewer samples per samples prior to
(wet) oxidizing agent) are blanks to correct for any regular wet run than standard wet mineral analysis
used to oxidize and organic matter in acid ashing. Minerals or dry ashing by rapid or official
incinerate organic and oxidizing agent stay in solution. procedures methods
matter, leaving Little or no
inorganic matter volatilization

16.4 SUMMARY equipped with exhaust filters, may be used without a fume
hood. Ashing may eventually be replaced by methods allow-
The two major types of ashing analysis, dry ashing and wet ing for the direct determination of mineral identity and con-
ashing, are used to determine total mineral content by con- tent from fresh samples. Due to its relative simplicity and
ventional means or by the use of microwave systems. The cost-effectiveness, however, ashing remains an essential
procedure of choice depends on subsequent analyses to be component of proximate analysis and is a key preparatory
performed and on cost, time, and sample number limitations. step for specific mineral analyses.
Conventional dry ashing is based on sample incineration at
high temperatures in a muffle furnace. Except for certain
volatile elements, which may be lost during dry ashing, the 16.5 STUDY QUESTIONS
residue may be used for both proximate analysis and further
specific mineral analyses. Conventional wet ashing is often 1. Identify four potential sources of error in the preparation
used as a preparatory step for specific elemental analysis, by of samples for ash analysis. Describe a way to overcome
the spectroscopy methods described in Chap. 9, since it each error.
simultaneously dissolves minerals and removes organic 2. You are determining the total ash content of a product
material via oxidation. Wet ashing conserves volatile ele- using the conventional dry ashing method. Your boss asks
ments but requires more dedicated operator time than dry you to switch to a conventional wet ashing method
ashing if the process is not automated, is limited to a smaller because they have heard that it takes less time than dry
number of samples, and requires the use of highly caustic ashing.
solvents. Microwave ashing (dry or wet) is faster than con- (a) Do you agree or disagree with your boss concerning
ventional methods and requires little dedicated space, and if the time issue and why?
16 Ash Analysis 269

(b) If fast sample throughput is the primary concern and 2. A sweet potato sample (23.5000 g) was found to have
cost is less of an issue, what two methods might you 0.0850 acid-soluble ash and 0.0940 g acid-insoluble ash.
suggest? What is the percentage acid-insoluble ash?
(c) Not considering the time issues, why might you want 3. You wish to have at least 100 mg of ash from a sample of
to continue using dry ashing, and why might you oats. Assuming 2.5% ash on average and no sample loss,
change to wet ashing? what is the minimum weight of oats required for ashing?
3. Your lab technician was to determine the ash content of 4. You wish to have a coefficient of variation (CV) below
yogurt by conventional dry ashing. The technician 5% with your ash analyses. The following ash data are
weighed 5 g of yogurt into one preweighed platinum cru- obtained: 2.15%, 2.12%, and 2.07%. Are these data
cible, immediately put the crucible into the muffle fur- acceptable, and what is the CV?
nace using a pair of all stainless steel tongs, and ashed the 5. A sample of a plant-based protein burger was analyzed.
sample for 48 h at 800 °C. The crucible was removed Here are the results: sample weight, 2.034 g; weight after
from the muffle furnace and set on a rack in the open until drying, 1.0781 g; weight after ether extraction of the
it was cool enough to reweigh. What errors were made in dried sample to remove fat, 0.4679 g; and weight of ash,
the preceding method? What were the likely results of 0.0233 g. What is the percentage of ash on (a) a wet
these errors? What instructions should you have given weight basis, (b) a fat-free basis (including water), and (c)
your technician before starting the procedure in order to a fat-free basis (without water)?
avoid the errors you noted?
4. How would you recommend to your technician to over- Answers
come the following problems that could arise in conven- 1. (a) 1.70%, (b) 1.92%
tional dry ashing of various foods? Calculate ash from sample:
(a) You seem to be getting volatilization of phosphorus Crucible + ash 28.5939 g
when you want to later determine the phosphorus Tared crucible 28.5053 g
content. Ash 0.0886 g
(b) You are getting incomplete combustion of a product
high in sugar after a typical dry ashing procedure (a) Calculate for ash on a wet weight basis (a):
(i.e., the ash is dark colored, not white or pale gray). 0.0886 g ash
× 100% = 1.70% or 1.7%
(c) You have reason to believe the compound you want 5.2146 g sample
to measure after dry ashing may be reacting with the
porcelain crucibles being used. (b) Calculate for ash on a dry weight basis (b):
(d) You want to determine the iron content of some foods 0.0886 g ash
but cannot seem to get the iron solubilized after the  100% − 11.5% 
5.2146 g sample ×  dry matter coeff . 
dry ashing procedure.  100% 
5. Identify the advantages and disadvantages of using wet ×100% = 1.92%
and dry microwave ashing compared with conventional or
wet and dry ashing. Consider initial investments needed
for equipment, the cost of supplies and reagents, as well 11.5 g water
as the relative energy costs, time invested to prepare, run, 5.214 g sample × = 0.5997 g water
100 g sample
and clean up after analyses, and the use and disposal of
hazardous chemicals. 5.214 g sample − 0.5997 g water = 4.6149 g sample dry wt

0.0886 g ash
× 100% = 1.92%
16.6 PRACTICE PROBLEMS 4.6149 g dry wt sample

1. A green coffee sample was found to contain 11.5% mois- 2. 0.4%

ture. A 5.2146-g sample was placed into a crucible Calculate % insoluble ash:
(28.5053 g tare weight). The ashed crucible weighed
0.0940 g acid insoluble ash
28.5939 g. Calculate the percentage ash on (a) an as-­ × 100% = 0.4%
received (aka. fresh or wet weight) basis and (b) on a dry 23.5 g sample
matter basis. Make sure to adjust for significant figures in The content of acid-soluble ash is irrelevant to the cal-
this and other practice problems. culation of insoluble ash percent.
270 G. K. Harris

3. 4 g (c) Calculate on fat-free, dry weight basis:

100 mg = 0.1 g ash 0.0233 g ash
2.5% = 2.5 g ash/100g sample × 100%
( 0.4679 g dry sample wt without fat )
2.5 g ash 0.1 g ash = 4.98% ash , fat-free, dry basis
=
100 g sample x

2.5 x = 10
Acknowledgments The author of this chapter and the book editors
x = 4 g sample thank Maurice R. Marshall (Department of FoodScience and Human
Nutrition, University of Florida, Gainesville, FL, USA) for his contri-
bution to the chapter in the previous edition of the book. Thanks is also
4. Yes, 1.9% expressed to Bob Lockerman and Eileen Stochl of the CEM Corporation
for providing microwave ashing equipment images and checking rele-
Calculate the mean:
vant chapter text. The author of this chapter recognizes and thanks Ruth
2.15% + 2.12% + 2.07% Watkins (Department of Food, Bioprocessing and Nutrition Sciences)
= 2.11%
3 for contributions to the current chapter. A special thanks is given to
students, who encourage us to think more deeply about each topic we
Calculation of mean and standard deviation was done research and teach.
using Excel:
1 2.15%
2 2.12%
3 2.07% REFERENCES
Average = 2.11%
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SD
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x 2. Akinyele, IO, Shokunbi, OS (2015) Comparative analysis of dry
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2.11
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Sample wet wt 2.034 g the beginning student to follow.
4. ASTM (American Society for Testing and Materials) D4309-96
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0.0233 g ash are discussed under each major heading.
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16 Ash Analysis 271

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