Analysis of the Effect of Alkaline Hydrolysis Cremation on
Minerals and Trace Metals in Bone
Senior Honors Thesis
Rebekah Quickel
Chemistry and Physics Department
California University of Pennsylvania
May 11, 2017

2
Abstract:
This research seeks to determine the changes that occur in bone during the
Alkaline Hydrolysis Cremation (AHC) process. AHC is a form of cremation which is
considered by many to be an environmentally friendly alternative to traditional fire
cremation. AHC is typically performed in a strongly basic solution under increased
pressure and temperature to accelerate the natural decomposition a body typically
undergoes. This process results in human or animal remains being completely
decomposed with only bone ash remaining, which can be returned to the family. The
AHC process was performed using pig femur bones in a pressure cooker with a
potassium hydroxide solution to mimic the commercial process. Bone structure was
analyzed qualitatively by visual inspection of the bones and quantitatively by monitoring
the concentrations of calcium, magnesium, and iron ions during the AHC process. Metal
ion analysis was performed using atomic absorption spectroscopy. The concentration of
calcium fell within the range of 30.3 ppm to 125.0 ppm, the iron concentration ranged
from 17.5 ppm to 68.0 ppm, and the magnesium concentration ranged from 7.65 ppm to
24.3 ppm.

1
Introduction:
For as long as humans have been alive, they have also been dying. Some of the
earliest cases of ritual burial of the dead can be found at sites occupied by Homo
neanderthalensis, or Neanderthals, over 50,000 years ago.1 This form of symbolic burial
has never been seen at other sites of known prehistoric occupation and shows it to be a
human specific trait. The practice of burial as a funerary process did not end with the
Neanderthal extinction. Burial is the most popular form of funerary practice in the United
States and as of 2005, over 61.4% of deceased individuals were buried.2 Typical burials
involve the deceased individual being placed in a coffin or other vessel and being placed
in the ground. Often the site is marked with a headstone or marker so that others do not
accidentally disturb the remains by not knowing they are present. While burial remains
the most common form of funerary practice in the United States, preferences are
changing. Over the years, new techniques have been developed and used as an alternative
funerary process for those opposed to burial.
The second most popular funerary practice in the United States is traditional fire
cremation. This technique breaks down the deceased so that the residual remains can be
returned to the family. The body is broken down in a furnace where it undergoes a
combustion reaction with natural gas and the body as the fuel sources. After the reaction
goes to completion, the bones are still mostly solid and must be crushed into bone ash, or
small, broken down particles of bone, to be returned to the family.3 Over the past years,
fire cremation has steadily become a more favorable funerary practice in the United
States, with a projected 58% of individuals being cremated in 2020, up from 32.3% in
2005.2 The reasoning behind the switch from traditional burials to cremation can be due

2
to many factors, including religious and personal reasons. The lower cost for cremation
over burial, a difference of over $1,000, can also be the deciding factor for many
families.4
More recently, a new movement for environmentally friendly funerary practices
has led individuals to improve on the traditional burial and cremation practices already
widely used in the United States and around the world. Traditional fire cremations release
a large amount of carbon dioxide gas into the atmosphere and any plastics intentionally or
inadvertently left in the casket before cremation end up being vaporized to produce toxic
fumes that are also released into the environment. Medical implants, such as hip and knee
replacements or pacemakers, and prosthetics must be removed from the deceased before
cremation as they can contain batteries which can lead to explosive ruptures when
exposed to extreme temperatures, such as those created during the cremation process.5
Removal of such implants can incur additional costs for the family depending on their
situation.
In order to find a “greener” treatment of humans remains, the alkaline hydrolysis
cremation (AHC) process was developed. This newer technique has been in use since the
1990’s by many research universities to dispose of donated corpses and deceased animals
used in disease research. This form of cremation uses an alkaline hydrolysis reaction of
aqueous solutions of potassium hydroxide or sodium hydroxide to break down the body
into its basic, soluble, organic components and bone ash. At room temperature and
pressure, this reaction can take up to twelve hours to fully decompose a human body;
when the temperature and pressure are raised, the reaction rate increases to the point
where a whole human body will decompose in just three hours.6 A typical AHC retort can

3
be seen in Figure 1 below. The machine has a similar style to a traditional cremation
retort except for the heating coils in place of the flame ignition source and an area for the
alkali solution to be introduced to the chamber. The flesh of the body is first impacted by
the alkali solution, causing the cells to rupture and begin breaking down into smaller
organic molecules. Once the flesh is dissolved, the solution attacks the bone.

Figure 1: AHC retort/machine used to contain the remains and alkaline solution.
The machine allows for a specific temperature and pressure to be achieved for
efficient cremation.7

Figure 2: Image on the left is a form of bone ash that remains after completion of
alkaline hydrolysis cremation. Although the bone is already in extremely small
pieces, it can be further crushed to be returned to the family.8 Image on the right is
crushed bone resulting from this experiment, after the AHC process and dried.

4
The remaining bone, which can be seen in Figure 2 above, is brittle and soft
which allows the bone to be further crushed, if necessary, to be returned to the family.
The end result is an organic solution with a basic pH that can be neutralized for use as
fertilizer in gardens or other allowed areas. While the entire process requires heating, the
carbon dioxide emissions created during the reaction are minimal when compared to fire
cremation, and pose little threat to the environment. Medical implants and prosthetics do
not have to be removed since they are not harmed during the reaction and can be removed
from the bones afterwards, reducing costs for their removal.
This technique can also destroy infectious disease precursors, which are difficult
to destroy using an incinerator. It was found that prion proteins can be inactivated using
sodium hydroxide with a detergent, such as sarkosyl, and is typically used as a foaming
agent in cleaning products. The detergent strips the outer layer of the prion protein,
giving the base more surface area to react and breakdown the prion into amino acids and
other small molecules.9 This makes AHC the ideal technique for disposal of infectious
waste and animal carcasses used for research purposes.
Even though the immediate reaction between the body and the alkaline solution is
the breakdown of the flesh and organic tissue, when the flesh is gone, the base reacts with
the skeleton, which is much harder to dissolve. Bone is composed of a number of organic
and inorganic components, contributing to the strength and stability of bone which causes
it to be difficult to break down.

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Figure 3: Cross section of the right proximal femur. The dense bone covers the
outer surface of the bone and thickens as it moves downward from the femoral
head. The spongy bone remains in the head and greater trochanter and extends to
the marrow channel.11
The densest bones in the human body are long bones; the ones found in the limbs.
As seen above in Figure 3, the general composition of bone is a thick layer of cortical or
dense bone that gives the outward appearance and solidity of the bone. At the proximal
and distal ends are lighter portions of bone called trabecular or spongy bone. This light
and airy portion of bone has a lattice work appearance and has many vacancies. These
open areas allow the spongy bone to absorb impact forces in high impact areas of the
body, such as the joints and balls of the feet. Without the spongy bone cushioning the
ends of the bones, the dense bone would fracture under much less force.10

Figure 4: Summary of femoral bone landmarks, head, greater trochanter, and
condyles, and direction. Proximal refers to the upper half of the femur while the distal
end refers to the lower half of the bone.

6
The areas of spongy bone present in the long bones correspond to the locations of
the epiphyses, or growth plates in long bones. In Figure 4 above, there are three areas
corresponding to the growth plates, the condyles, femoral head, and greater trochanter.
These three sections of the bone are not fused to the femur shaft at birth, rather they fuse
later in life, between 15 to 20 years old, which makes growth plate fusion a useful
indicator for age estimation. The condyles lie on the distal portion of the femur, or closer
to the knee, while the head and greater trochanter lie on the proximal end, closer to the
pelvis.
The microscopic organization of the dense bone includes osteons, allowing for
blood vessels which feed the bone and marrow, to pass through to the bone center. Those
blood vessels also feed osteocytes residing within the osteon.12 Osteocytes control the
formation and removal of bone though the signaling of osteoblast and osteoclast cells.
The osteoblast cells build up bone, while osteoclasts cause bone resorption. Osteocytes
are most important in bone repair after a break or fracture due to their job in signaling
bone healing. The organization of these osteons and osteocytes can be seen below in
Figure 5 in the cross section of a long bone. When the osteocytes stop functioning
properly, due to injury or deformation, the bone strength and structure can be affected by
deformation or loss of mineral components.10

7

Figure 5: Breakdown of bone structure showing the collection of osteons and
osteocytes.13
While the osteons and osteocytes give bone its structure, the strength of the bone
comes from its organic and inorganic material. The largest organic component present in
every human and animal bone is collagen, a large protein molecule that gives bone its
flexibility.14 At birth, bone is composed entirely of collagen fibers, which intertwine to
form the bone structure. As the individual and bone ages, the collagen fibers begin to be
replaced with a hard, dense, inorganic mineral called hydroxyapatite.
Hydroxyapatite, Ca10(PO4)6(OH)2, is a member of the apatite mineral family
whose main component is calcium phosphate.15 In early bone development, pockets of
hydroxyapatite are stored within the collagen fibers as they are created. As the bone ages,
the hydroxyapatite mineral covers the collagen fibers and creates the dense bone structure
that is most commonly seen.14 This mineral is the main component used in bone
remodeling and is synthesized by the osteoblasts to heal fractures or add material for
bone growth. It can also be broken down by the osteoclast cells when mineral needs to be
removed from the bone surface.15

8

Figure 6: Crystal structure of calcium phosphate mineral with a hexagonal unit
cell.17, 18
The crystal structure of hydroxyapatite, seen in Figure 6, is only 50 nm in size
with lattice parameters of a-9.436 Å and c-6.882 Å. Groups of phosphates and alcohols
bound to their respective hydrogens are shown.16 However, all the groups, including the
calcium ions, are not bound to each other. This allows all three components of
hydroxyapatite to be exchangeable for other ions or groups. The ion and group exchanges
change the function of the hydroxyapatite in that particular spot and can cause it to build
or destroy bone. Some of the exchanges that are possible are magnesium, zinc, strontium,
and iron for calcium, carbonate groups for the phosphates, and also fluorine for the
alcohol groups.15
The metal exchanges in the crystal structure have a large impact on the mineral
and bone structure. Strontium has been seen to stabilize the bone structure and also
stimulates bone formation and works to prevent bone loss. On the other hand, zinc has
been found to help maintain healthy bone and is vital to protein synthesis within bone.15
Both of these metals contribute to changes within bone, but neither help with bone
strength and stabilization. Magnesium and iron have been found to influence both of
those characteristics. Iron is essential for collagen synthesis, keeping the bone slightly

9
flexible, so it does not break when force is applied. It can also prevent bone resorption
helping to increase the strength of bone by preventing bone mass loss and boosting
hydroxyapatite synthesis.15 Magnesium, on the other hand, controls osteoblast and
osteoclast activity through signaling. The magnesium ions can reside on the surface of the
hydroxyapatite or within the apatite structure, which increases the strength of the bone.19
The research presented here focuses on the alkaline hydrolysis reaction with bone.
While the use of an acidic solution as the reactant with bone is typically considered for
disposal, it removes the hydroxyapatite mineral from bone and leaves the collagen
behind. This results in a flexible and bendable bone being left over, which is difficult to
further break down.14 The use of a basic solution with bone employs a reaction like that
shown below in Figure 7.

Figure 7: Alkaline hydrolysis reaction between potassium hydroxide and calcium
ions from the hydroxyapatite bone structure.20
In this reaction, the potassium hydroxide or sodium hydroxide, depending on the
experimental conditions, will dissociate into aqueous K+ and OH-. The OH- will react and
bind with the Ca2+ within the hydroxyapatite structure in a 2:1 ratio of hydroxide ions to
calcium ions. The resulting calcium hydroxide will form a solid and precipitate out of the
basic solution, effectively removing the calcium from the crystal lattice structure and
leaving a vacancy in its place. The potassium ions can also bond with the PO43- groups, in
a 3:1 ratio, creating soluble K3PO4. Solubilizing the phosphate could further destabilize
the hydroxyapatite structure.

10
Theoretically, this calcium ion vacancy should result in the degradation of the
bone structure and lead to the bone ash that remains after the AHC process is complete.
Some research suggests that the vacancies left behind by the calcium being removed
allows other cations to fill the vacancies, thus changing the bone characteristics. In the
reaction above in Figure 7, the K+ would fill those Ca2+ vacancies and not cause the bone
to degrade. Instead, it is possible that the K+, or other cations, could make the bone
structure stronger, based on the type of ion that takes the place of calcium.20
Nevertheless, for the AHC process to successfully decompose a human or animal carcass,
something must change within the bone structure that causes it to break down. It is
possible that the removal of the calcium ions creates so many vacancies that only a few of
the vacancies can be filled and, therefore, the unfilled vacancies still cause the bone to
breakdown during the AHC process.
Another way to monitor the bone decomposition during AHC is to track the
removal of ions from the bone. This can be done by detecting the concentration of the
metal ions present in the resulting reaction solution. Since calcium can be removed from
the bone and cause it to begin to break apart, the removal of other metals, such as
magnesium and iron, that are essential to bone structure and strength, could also occur.
Since magnesium can reside within the hydroxyapatite structure and on its surface, the
presence of magnesium can be an indicator that the hydroxyapatite structure is beginning
to decompose since magnesium is being released.19 The same can be done with iron,
when iron is detected in the reaction solution the bone structure is breaking down.
A useful method for detection of metals in solution is atomic absorption
spectroscopy. Atomic absorption spectroscopy converts a solution sample into an atomic

11
gas by burning off the rest of the solution using a flame or other thermal source. The
atoms are then bombarded with monochromatic photons and the amount of light absorbed
by the atomized sample is detected. By using the absorbance and a Beer’s Law plot of
absorbance vs. concentration, the concentration of the sample can be determined. A
plethora of metals can be detected using atomic absorption spectroscopy with high
accuracy.21
The objectives of this research were to first successfully cremate a pig femur
bone, then to identify the changes that occur to the bone structure during the AHC
process by monitoring the change in calcium, iron, and magnesium ions being removed
from the bone throughout the AHC process.
Procedure:
This experimental procedure was adapted from the procedure by Wang and
associates,22 which looked to produce the optimal conditions to perform AHC. A Presto
01370 8-Qt Stainless Steel pressure cooker was obtained and fitted with a dual
temperature/pressure gauge to monitor temperature and pressure changes during the
experiment. A Durabrand Single Burner 1100 watts Variable Temperature Control
heating element (WS 100) was used for the heating element. The set-up can be seen in
Figure 8 below.
Pig bones were used in place of human bones for this experiment since pigs are
often used as a suitable model for humans in research. Four femur bones were obtained
from Cheplic Meat Processing and analyzed for trauma due to the butchering process.
Each bone was weighed prior to cremation and placed in the middle of the pressure

12
cooker. A 2 liter solution of 0.89 M potassium hydroxide (KOH) was added to the
pressure cooker and filled with an additional 1.5 liters of deionized water to fill the
pressure cooker halfway and prevent the cooker from running dry. The bone set up can be
seen in Figure 9 below. The pressure cooker was heated for a total of three hours and
depressurized every thirty minutes to remove a sample for later analysis using atomic
absorption spectroscopy.

Figure 8: Pressure cookers
set up with attached gauge
and heating element.

Figure 9: Placement of pig
femur bone, filled halfway
with 3.5 L KOH/H2O.

Bone Trauma:
Each of the four pig femurs was received with noticeable signs of trauma caused
by the butchering process. Any trauma to the bone before cremation began could have
impacted the length of time and extent of cremation. Any impact to the cartilage
surrounding the bone should have no impact on cremation, while deeper trauma
impacting the trabecular of cortical bone will introduce an additional entrance point for
the KOH solution and speed up the cremation process. All femurs were sided, weighed,
and measured for femur length, head diameter, and diameter at mid-shaft and can be seen
in Table 1 below. All trauma was recorded for future analysis and can be seen in Table 2

13
and images of the trauma can be seen in Appendix A. All femurs appeared to have fused
epiphyses, indicating an adult pig.

Femur
Designation
1
2
3
4

Side

Weight
(g)

Femur
Length
(cm)

Head
Diameter
(mm)

Diameter at
Mid-Shaft
(cm)

Right 373.81
19.6
38.0
8.9
Left 472.43
20.4
37.2
10.4
Left 488.93
20.5
41.5
10.4
Left 453.66
20.3
37.0
9.3
Table 1: Femur sides, weights, and measurements.

Femur
Designation

Trauma

Slice to the posterior, medial condyle impacting trabecular bone.
Superficial slice to medial surface of head, extending slightly to the
1
neck.
Slight incision of the shaft, superior to the patellar surface, lateral side.
Superficial slice to medial surface of the femoral head, impacting
2
cartilage.
Slight shave off of condyle, more so on the medial side than lateral
3
side, impacts trabecular bone.
Slight laceration on head extending to the neck.
4
Scrape off the top head, exposing trabecular surface.
Table 2: Trauma recorded for each femur bone prior to cremation.
Atomic Absorption Spectroscopy:
All samples were analyzed using a Shimadzu AA-7000 atomic absorption
spectrometer with and ASC-7000 Shimadzu auto sampler. To analyze the AHC samples
by atomic absorption (AA) spectroscopy, the samples required acidification prior to
dilution. A 5 mL portion of the collected sample was mixed with 5 mL of 0.8 M HNO3 in
a 10 mL volumetric flask. The samples then had to be filtered using gravity filtration due
to a brown foam forming with a peroxide odor on the surface of the liquid sample. From
the 10 mL sample, one mL was removed and diluted with deionized water in a 25 mL
volumetric flask. The 25 mL sample were used for AA analysis.

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Results:
A total of three cremations were performed and each will be discussed below.
Femur 1:
This femur was the smallest bone of the four and was used as an initial trial to test
the experimental set-up and procedure before the rest of the bones were tested. Using the
smallest femur also eliminated femur size and weight as a variable to influence extent of
cremation. As described above in the procedure, the pressure cooker was only filled with
2 liters of 0.89 M KOH solution as outlined by Wang and associates22 and left
undisturbed for three hours to determine what affect depressurizing the pressure cooker
will have on the extent of cremation. After two and a half hours had elapsed, the steady
stream of pressure coming from the release valve was interrupted and a yellow substance
began to spew out the valve. The heat was immediately turned off and the cooker
depressurized. The result of the first trial can be seen in Figure 10 below.

Figure 10: Result of yellow substance spewing from the release valve of the pressure
cooker, coating the lid and fume hood sash in the substance.

15
After the lid was removed, the pressure cooker revealed a dark red solution along
with the bone. Even though the cremation did not proceed for the full three hours, the
resulting bone still showed signs of breakdown and can be seen in Figure 11 below.

(A)

(B)

(C)
(D)
Figure 11: (A) remaining KOH solution and femur broken into three pieces. (B)
main femur shaft with proximal and distal ends missing and slight red discoloration.
(C) distal portion of femur, condyles, partially crushed. (D) proximal portion,
femoral head.
Femur 2:
In response to the results of femur 1, an additional 1.5 liters of deionized water
was added to fill the pressure cooker halfway to prevent the pot from running dry. A total
of 30 mL of the KOH/H2O sample were taken every thirty minutes for atomic absorption
analysis and the water was replenished to the half-way mark with approximately 100 to
300 mL of deionized water in the pressure cooker after sample removal. Depressurization

16
began five minutes before each sample was taken. At the end of three hours, the solution
was filtered to catch any bone particles and to retrieve the larger bone parts which can be
seen below in Figure 12.

(B)

(A)

(C)

(D)

(E)
(F)
Figure 12: (A) femur broken into four parts. (B) relative placement of each
epiphyseal surface to the femoral shaft and discoloration. (C) cracking and
discoloration of distal femur. (D) cracking of epiphyseal surface of the medial
condyle with discoloration. (E) greater trochanter with exposed spongy bone and
discoloration. (F) femoral head showing discoloration halting at epiphyseal surface.

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Atomic absorption (AA) spectroscopy was used to analyze a total of six sample
solutions for calcium concentration of the pressure cooker solution. Figure 13 below
shows the standard curve created for sample analysis. The curve ranged from
0.5 to 5 ppm with an extremely high linearity of 0.9979. Table 3 below shows the
absorbance of each sample and their corresponding concentrations back-calculated to the
original pressure cooker environment.

Beer's law plot for calcium
0.45
0.4

Absorbance

0.35

y = 0.0803x - 0.0028
R² = 0.9979

0.3
0.25
0.2
0.15
0.1
0.05
0
0

1

2

3

4

5

6

Concentration (ppm)

Figure 13: Beer’s law standard curve created for calcium. The concentration of each
solution was calculated using the linear regression equation in the graph above.
Femur 2 calcium concentration
Concentration in
Collection
Concentration
Abs.
original solution
Time (min)
(ppm)
(ppm)
0.0486
0.640
560.1
30
0.0575
0.751
651.4
60
0.0597
0.778
669.4
90
0.0575
0.751
640.2
120
0.0516
0.677
730.8
150
0.0546
0.715
805.1
180
Table 3: Summation of sample absorbance and concentrations to original solution.
Femur 3:

18
No cremation was conducted on this femur due to the bone having decomposed
too far since it was left too long in the refrigerator instead of freezer before it was used.
The femur was eliminated to prevent the natural decomposition process from influencing
the AHC process being performed.
Femur 4:
In an effort to fully cremate the bone, the cremation time was raised to four hours
instead of three hours. The bone was partially frozen, inhibiting decomposition that
occurred in femur 3, and was thawed fully before cremation. Two additional samples
were extracted at 3 hrs and 30 minutes and 4 hrs and a third sample was added to the
beginning at 0 minutes. Instead of removing 30 mL of sample, only 20 mL of sample
were removed and 100 to 300 mL of deionized water was once again added to the
halfway mark. The time zero sample was removed when the bone was placed in the
KOH/H2O solution and prior to heating and pressurization. Depressurization began five
minutes before each sample was taken. During the third hour, banging noise was heard
within the pressure cooker due to the bone bouncing around in the solution after
additional water was added. The solution was filtered before the bones were removed.
The bone results can be seen in Figure 14 below.

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(A)

(B)

(C)
(D)
Figure 14: (A) remains of femur 4 after four hours of cremation with discoloration
seen on the majority of the bones. (B) front view of distal femur showing extreme
cracking and discoloration. (C) view of epiphyseal surface showing minimal
discoloration and extensive cracking of sides. (D) remains of the femoral head,
greater trochanter, and condyles.
Femur 4 was also left to dry completely to determine its brittleness and ease at
which it could be crushed. The bone was left to air dry for five days and was examined
for any changes to the color and structural integrity of the bone. Images of the bone can
be seen in Figure 15 below.

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(A)

(C)

(E)

(B)

(D)

(F)

(G)
(H)
Figure 15: (A) remains of bones after drying for five days. (B) exposure of spongy
and trabecular bone on the medial surface of the distal femur. (C) extended
cracking of frontal femur. (D) bone peeling on the sides of the epiphyseal surface.
(E) appearance of crack and gouge on the femoral neck. (F) crack extending
downward on proximal femur. (G) empty marrow chamber. (H) remains of fully
crushed femur 4.

21
Analysis of concentrations of calcium, iron, and magnesium was conducted by
AA. The samples were acidified as stated in the experimental section in order to perform
the analysis. Three standard curves were created to calculate the concentrations of the
metals based on their specific absorbance. The curves and their corresponding equations
can be seen in Figure 16 below. Iron was measured in the range of 0.1 to 0.5 ppm and
magnesium ranged from 0.05 to 0.1 ppm with a high linearity of 0.9964 and 0.9933
respectively. Tables 4 through 6 display the absorbance and calculated concentration of
each metal.

Beer's law plot of calcium, iron, and
magnesium
1.4

Absorbance

Calcium

y = 12.47x + 0.0295
R² = 0.9933

1.2
1

Iron

y = 0.7988x - 0.0014
R² = 1

0.8
0.6

Magnesium

0.4
y = 0.0396x + 0.0006
R² = 0.9964

0.2
0
0

0.2

0.4

0.6

0.8

Linear (Calcium)
1

Linear (Iron)

Concentration (ppm)

Figure 16: Combined standard curves for calcium, iron, and magnesium with
corresponding linear regression equations used to calculate the concentration of each
metal sample.

22
Femur 4 calcium concentration
Concentration in
Collection
Concentration
Abs.
original solution
Time (min)
(ppm)
(ppm)
0.0568
0.0729
63.8
0
0.0264
0.0348
30.3
30
0.0369
0.0479
41.5
60
0.0336
0.0438
38.8
90
0.0359
0.0467
44.8
120
0.0330
0.0431
42.6
150
0.0507
0.0652
68.5
180
0.0313
0.0409
45.9
210
0.0837
0.1065
125.0
240
Table 4: Absorbance and corresponding calculated calcium concentrations in the
sample and original pressure cooker solution.
Femur 4 iron concentration
Collection
Concentration in
Concentration
Time
Abs.
original solution
(ppm)
(min)
(ppm)
0.0020
0.0354
30.9
0
0.0014
0.0202
17.6
30
0.0014
0.0202
17.5
60
0.0018
0.0303
26.8
90
0.0023
0.0429
41.2
120
0.0028
0.0556
54.9
150
0.0028
0.0556
58.3
180
0.0030
0.0606
68.0
210
0.0019
0.0328
38.5
240
Table 5: Absorbance and corresponding calculated iron concentrations in the
sample and original pressure cooker solution.

23
Femur 4 magnesium concentration
Collection
Concentration in
Concentration
Time
Abs.
original solution
(ppm)
(min)
(ppm)
0.179
0.0120
10.5
0
0.180
0.0121
10.5
30
0.379
0.0281
24.3
60
0.297
0.0214
19.0
90
0.238
0.0167
16.0
120
0.188
0.0127
12.6
150
0.104
0.00597
6.27
180
0.115
0.00682
7.65
210
0.182
0.0122
14.3
240
Table 6: Absorbance and corresponding calculated magnesium concentrations in the
sample and original pressure cooker solution.
Discussion:
Femur 1:
As seen in Figure 11 (A), it appeared that all of the water had been cooked out of
the solution. The resulting liquid would have consisted of KOH, collagen, and other
organic substances which began to turn to a gelatinous substance that began to foam and
exit through the release valve. The resulting bone revealed some interesting
characteristics. The femur can be seen to have detached into three different parts, as seen
in Figure 11 (A). The detachment points coincide with the epiphyseal or growth plate
areas of the femur. Upon first inspection of the femur, it appeared to have fused
epiphyses, meaning the condyles and head should not have detached as easily since they
would have become one solid bone with the femoral shaft. This suggests that the pig was
much younger than first thought, between two to three years at death.
In Figure 11 (B-D) the red discoloration is due to the bones sitting in the
concentrated solution. The breakdown of the condyles in (C) is evidence of cremation.

24
The bone was fragile to pick up and the removal of the bone from the pot resulted in the
damage. The edges of the proximal and distal femur in Figure 11 (B) as well as the head
in (D) had a softer feel when compared to the femoral shaft which was still as firm as it
was before the cremation. The softer feel to the bone is further evidence of cremation
because those are the points where the hydroxyapatite crystal structure is weakest due to
calcium removal. In these areas there is a large amount of trabecular bone at the
epiphyses, increasing the surface area available for the KOH solution to react with.
Therefore, the areas of the epiphyses will breakdown much faster than the rest of the
bone.
Femur 2:
The cremation of femur 2 was performed for the full three hours since additional
water was added to the pressure cooker and it did not run dry. The bone again separated
into four different sections, the femoral head, greater trochanter, femoral shaft, and
condyles. One difference between femur 1 and 2 is a green discoloration seen on the
distal portion of the femur 2 shaft, as well as the condyles, greater trochanter, and outline
of the femoral head. The placement of the green color coincides with the epiphyseal sites
of the bone and is also where a large amount of blood vessels flow to provide blood to the
growth plate. Therefore, the green discoloration is due to the breakdown of the haemin in
the blood at the ends of the bone. Haemin breakdown is also known to transform into
biliverdin and bilirubin which is green in color.23
The bones also showed more extensive cracking and breakdown than femur 1. In
Figure 12 (C) cracks are present on the anterior surface of the distal portion of the femur.
Those cracks only extend as far as the green discoloration but were not present on

25
femur 1. In image (D), a large chunk of the condyle is crumbling and was able to be
pulled off of the bone itself. In images (E) and (F), no cracking is present but the edges of
the bone are ragged and spongy looking. This is due to the removal of the layer of dense
bone, exposing the spongy bone below. These two bones show evidence of the KOH
solution eating its way through the outer bone surface to begin impacting the softer, inner
bone. This was not seen in femur 1. Since the reaction conditions were not equal for
femur 1 and 2, there is no way to conclusively tell if the cracks and breakdown appeared
due to the extra thirty minutes the bone was submerged in the KOH/H2O solution. The
combination of the green discoloration and the appearance of the breakdown of the bone
can be used as indicators of the extent of cremation. The more discoloration of the bone
as well as ragged, broken edges, the farther cremated the bone is.
The AA data, in Table 3, revealed a steady increase in calcium concentration
throughout the 3 hours of cremation. This is consistent with the breakdown of the bone
discussed previously. The absorbance and concentration seen in columns two and three
correspond to the 25 mL diluted sample used in the AA analysis. The concentration in
original solution in column four is the calcium concentration in the pressure cooker
solution, including the water added after each sample was taken.
The slight decrease in calcium concentration between 90 and 120 minutes could
be due to a decrease in the amount of calcium being removed from the bone while the
sample collection removed the calcium from the solution. Another reason for this
decrease can be the formation of a Ca(OH)2 precipitate. The acidification process was
used to dissociate the calcium and hydroxide, releasing the calcium back into solution for
analysis. If the acidification did not release all of the calcium from the precipitate, a

26
lower absorbance, and therefore lower concentration of calcium, will be detected by the
AA. This can be resolved by using a higher concentration of HNO3 for acidification and
will increase the amount of Ca(OH)2 that is dissociated.
Femur 4:
In an attempt to fully cremate femur 4, the cremation time was increased to 4
hours instead of 3. This resulted in the bone breakdown seen in Figure 14. The bone was
broken into significantly more pieces during this cremation. The femur shaft remained
intact but the condyles were completely destroyed, the remnants can be seen as the pile of
bone in image (A and D). This was most likely due to the bone being thrown around in
the solution and hitting other bone, as well as the sides of the pressure cooker, causing the
already brittle bone to break apart much more easily. Similar evidence of bone
breakdown was present on femur 4, as with femur 2, except it was more extensive in
femur 4. In image (A) the discoloration covers all of the bones, with a small patch of the
femur remaining a white color. Since the bone was in the KOH/H2O solution an
additional hour, the blood within the bone was able to breakdown further in femur 4 than
in femur 2.
In image (B) the discoloration is more concentrated in the lower half of the femur
and the cracking appears almost identical to the cracks in femur 2, which extends to the
edge of the discoloration. The cracking extends to the epiphyseal surface, which can be
seen in image (C). The bone appears to be peeling away from the main shaft, extending
laterally down the surface. All of the bones were extremely fragile and difficult to
remove from the pressure cooker without causing additional damage. Unique to this bone
was the condition of the femoral shaft. In femur 2, the shaft was very strong after three

27
hours and appeared to not have been greatly impacted by the KOH/H2O solution. In
femur 4, indents could be made in the shaft with a fingernail after removal from the
solution. This is evidence of the solution breaching through the top layers of the
trabecular bone and beginning to break down the hydroxyapatite structure.
Femur 4 was left to dry for five days to observe the bone structure both wet and
dry. Those images can be seen in Figure 15. Image (A) shows all the bones fully dry. A
color change occurred during this time with the entire bone taking on a grey/tan coloring
as it dried. This could be due to any remaining KOH/H2O solution drying on the outside
of the bone while the inside remained pure white like in image (G). Images (B through D)
show the distal end of the femur where the condyles were attached. The cracking that was
previously present in the wet bone has become more prominent and bone appears to have
broken off in some areas. Image (C) shows the appearance of spongy bone in the lower
middle portion of the bone. This means the dense bone has broken down enough to leave
the more pores bone underneath exposed or the basic solution ate away at the dense bone
on top, leaving behind pockmarks on the surface to give the appearance of spongy bone.
In image (D) a chunk of the edge of the bone does appear to be missing, exposing
the spongy bone, but does not extend back far enough to account for all of the pockmarklike bone. Therefore, the KOH solution did breakdown a section of the dense bone but
had just begun to affect another section of the bone, leaving behind the pockmarks. A
similar situation can be seen in image (B) on the medial surface of the distal femur, a
strip of smooth, dense bone with sections of pockmarked bone on either side due to
breakdown by KOH.

28
The proximal end of the femur seen in image (E and F) show cracking not
previously present in the wet bone. The crack extends parallel to the femoral head on the
posterior surface of the bone. A second crack can be seen running perpendicular to the
femoral head on the anterior surface of the bone, extending to the greater trochanter. No
other damage appeared in that area. Both ends of the bone were extremely fragile, and
when squeezed, shattered with a little application of pressure, the results of which can be
seen in image (H). The only part of the bone that remained intact was the central shaft
seen in image (G). The central marrow channel was completely empty, meaning the
marrow liquefied and leached out of the bone sometime during the cremation process and
leaving behind the bone white coloring.
Full analysis of calcium, iron, and magnesium ion concentration was performed
for femur 4, resulting in Tables 4 through 6 seen earlier. The fourth column shows the
concentration of each metal in the original pressure cooker solution. A graph of
concentration vs. time can be seen in Figure 17 below, comparing the three metal ion
concentrations throughout the cremation process.

Concentration (ppm)

Metal concentration change during AHC
process
140
120
100
80
60
40
20
0

Calcium
Iron
Magnesium
0

30

60

90 120 150 180 210 240

Time (min)

Figure 17: Graph comparing the concentrations of the three metals analyzed by
atomic absorption spectroscopy.

29
The first trend that can be seen in the graph is the high initial concentration at 0
minutes. This can be due to the sample having been collected when the bone was placed
in the KOH/H2O solution and began to decompose the little amount of soft tissue that
remained on the bone. After 30 minutes, the calcium and iron levels began to climb
steadily until calcium decreased at 210 minutes and iron decreased at 240 minutes. This
shows both calcium and iron were steadily removed from the bone structure over the
course of the cremation, which could have been one component of the bone breakdown.
A comparison of the calcium concentrations between femur 2 and 4 shows that
the calcium was removed throughout the whole cremation processes but the final
concentrations were strikingly different. Femur 2 had a significantly higher calcium
concentration over the entire cremation process and had a continuous increase over the
full 3 hours. This can be due to the small difference in linear regression equations for
each of the calcium samples. Even though the concentrations of calcium do not match for
both femur bones, the trend still remains that calcium is removed throughout the
cremation process.
An interesting aspect of the iron concentration (Table 5, Figure 17) is a dramatic
increase in iron between 90 and 120 minutes. This is most likely the point where the bone
marrow was liquefied and removed from the marrow channel within the bone. Since bone
marrow is rich in blood, the breakdown of the hemoglobin would have released a large
amount of iron into the solution, increasing its concentration. After 120 minutes, the iron
levels began to rise slowly again.
The third metal investigated, magnesium, had a very different trend in
concentration. Instead of an increasing concentration line, the line slowly decreases over

30
time after a peak at 60 minutes. This could be due to the magnesium not residing within
the bone structure at all, but in the small amount of flesh present or even in the cartilage
that decomposed rather quickly when exposed to the KOH/H2O solution. This is the
opposite of what was expected since the literature suggested magnesium played a key
part in the stability of the bone crystal structure. Another reason for the low concentration
could be the sensitivity of the instrument coupled with the sample being too diluted. The
amount of magnesium in the diluted sample may have been within the sensitivity range of
the instrument, causing it to register minimal changes in concentration over time.
Future work on this topic includes using scanning electron microscopy to obtain a
visual representation of the inside of the bone structure before and after the cremation
process. These images could provide additional evidence to determine what happens to
the bone that causes it to breakdown. Additional cremations can also be performed for
shorter time ranges to create a standardization of the bone breakdown at each stage of the
cremation process as well as cremating fleshed specimens to see the variation in the
process when flesh is added. Further investigating the trend seen in the magnesium
sample can lend more insight into bone composition and structure. Research can also
expand into other disciplines. Forensic anthropologists can analyze the bones to see what
can be deciphered from them after cremation, environmental scientists can find ways to
safely dispose of the alkaline solution, and mortuary scientists can further explore its use
for full body cremations.
Conclusion:
Three pig femur bones were cremated using the alkaline hydrolysis cremation
process to varying degrees of success. The second femur had portions of bones which

31
were able to be crushed without much force but did not achieve the complete cremation
that was expected. The fourth femur reached full cremation where the bone, once dried,
was crushed using only a strong squeeze of a hand. The analysis of the calcium, iron, and
magnesium metals in the basic solution showed interesting results. The calcium and iron
concentrations steadily increased throughout the entire AHC process but they decreased
around the fourth hour of cremation. The results of magnesium were the opposite of what
was expected. The magnesium concentration reached a maximum peak within 60 minutes
of cremation and steadily decreased over the next three hours. This could be due to
magnesium not being used internally within the bone structure as the literature suggested.
The successful cremation of two femur bones in four hours shows the ability to
reduce the commercial process to a small scale apparatus that can be used in a lab setting.
The results of the metal analysis show that the removal of calcium and iron are just one
factor that contributes to bone breakdown.
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Appendix A: Trauma Analysis Pictures
Femur 1:

(A)
(B)
Figure 18: (A) Slice to the posterior medial condyle, impacting the trabecular bone
only. (B) Superficial slice to the medial surface of head, extending slightly to the
neck.

Femur 2:

(A)
(B)
Figure 19: (A) Superficial slice to the medial surface of the femoral head, impacting
the cartilage. (B) Slice to the posterior lateral condyle, impacting the trabecular
bone only.

34

Femur 3:

Figure 20: Slight shave off of the medial condyle, more so than the lateral condyle,
impacting the trabecular bone.

Femur 4:

(B)
(A)
Figure 21: (A) Slight laceration on the head, extending to the neck. (B) Scrape off
the top head exposing trabecular bone.