American Journal of Anal yt ical Chemistry, 2011, 2, 979-983
doi:10.4236/ajac.2011.28114 Published Online December 2011 (http://www.SciRP.org/journal/ajac)
Copyright © 2011 SciRes. AJAC
Optimization of Dry Ashing of Whole Blood Samples for
Trace Metal Analysis
Stefanie A. Bragg, Zi-Lin g X ue
Department of Chemistry, The University of Tennessee, Knoxville, USA
Received July 20, 2011; revised September 14, 2011; accepted October 3, 2011
Dry ashing is an established method. Ashing whole blood samples are, however, often difficult to carry out
with significant sample loss, and the procedure is not well documented. A new procedure has been devel-
oped and optimized to dry-ash whole blood samples for trace metal analyses. The procedure reduces both the
dry-ashing time by more than two thirds and sample loss. The ashed sample can be readily used in subse-
quent, simultaneous or individual analysis of several metals by ICP-OES, as demonstrated in the analysis of
a whole blood sample. The new procedure is simple, inexpensive, and faster than the established method.
Keywords: Dry Ashing, Blood Samples, Trace Metal Analysis
Elemental analysis is one of the mostly widely used
methods in chemical analysis, and much progress has
been made to analyze [1-3]. Ashing procedures have
long been used to pretreat complex samples such as bio-
logical tissues and fluids prior to elemental analysis
[4-13]. Dry ashing in particular has been a standard in
the analysis of many biological samples, and it has been
used as a benchmark in the development of new analytic-
cal methods [9-12,14-23] with many citations in this year
alone [15-20]. Other techniques have been used as well
but these are either expensive [4,6] or more ideal for
identifying specific elements [5,8,13,24]. Microwave di-
gestion has recently found use for pretreatment. But it
requires costly instrumentation and like wet digestion,
the sample size it handles is small, usually ten grams or
less [25,26]. In comparison to wet or microwave diges-
tion, dry ashing is an inexpensive method that allows for
multiple elemental analyses in one sample without the
addition of chemical interferrents. In particular, dry ash-
ing allows occasional users to analyze biological samples
without the use of costly instrumentation. Its most attrac-
tive feature, though, is the ability to preconcentrate sam-
ples for detection of trace metals.
During our recent studies of new analyses for trace
chromium in blood, we have found that while there is
still prevalent use of dry ashing for these samples there
are few guidelines to follow [9-12]. The lack of stan-
dardization and temperature monitoring for dry ashing of
blood samples leads to a long process (often several days)
with significant sample loss. We have developed a new
dry ashing process with the use of thermocouples. The
temperature of the process is elevated in a gradient man-
ner but without the need of a muffle furnace. This new,
optimized process significantly reduces the dry ashing
time and sample loss. By using an inductively coupled
plasma-optical emission spectroscopy (ICP-OES) ana-
lyzer, we have used the dry ashing for the analyses of
trace metals in blood samples. The development of this
new process for dry ashing of whole blood samples is
Ceramic evaporating dishes, Pyrex watch glasses, high
purity grade nitric acid, potassium oxalate, copper AA and
zinc AA standards were purchased from Fisher Scientific,
Pittsburgh, PA. Chromium and iron AA standards were
purchased from Sigma-Aldrich, St. Louis, MO. Vanadium
AA standard was purchased from Ricca Chemical Com-
pany, Arlington, TX. Molybdenum AA standard was pur-
chased from High-Purity Standards, Charleston, SC.
Whole blood samples (porcine) were obtained from
Wampler’s Farm Sausage, Lenoir City, TN. Samples were
collected in Nalgene bottles using potassium oxalate as
anti-coagulant. Trace metal detection was performed
using a Perkin Elmer ICP-OES Optima 2100 DV.
S. A. BRAGG ET AL.
2.1. Method A (New Procedure)
Whole blood (40.1 - 71.3 g) was placed in a ceramic
evaporating dish, covered with a watch glass, and
placed on a hot plate at 80˚C - 100˚C in order to
evaporate water in the sample slowly without bumping
or boiling. Upon evaporation of water, the dish was
transferred to a Bunsen burner and heated slowly with a
cool flame (250˚C - 300˚C) initially in order to avoid
boiling and bumping. After 30 min, the temperature of
the flame was increased to 450˚C and the sample was
heated continuously for several hours. Intermittent stir-
ring allowed even ashing and monitoring of the sample.
Ashing was determined complete when the sample was
a rust red-brown color, typical for iron oxides, and no
black substance, indicating carbon, remained [21,22].
Total ashing time was dependent on the volume of
sample in the evaporating dish.
Standard additions for the detection of chromium by
ICP-OES was used in the current work. Thus this new
procedure was performed 5 times using 223 g of whole
blood in order to obtain sufficient ash (2.44 g).
2.2. Method B (Established Procedure) 
In the current work, the reported method was slightly
modified by using parallel steps as in the new method.
This is not the recommended procedure.
The following was conducted for comparison with the
new procedure (Method A). Whole blood (40.1 g) was
placed in a crucible and placed on a hot plate at 80˚C -
100˚C in order to evaporate water in the sample. After 8
h, the crucible was transferred to a Bunsen burner and
heated slowly with a cool flame. After 90 min, the tem-
perature of the flame was increased to 450˚C and the
sample was heated continuously for several hours. Ash-
ing was determined complete when the sample was a rust
red-brown color, typical for iron oxides, and no black
substance, indicating carbon, remained [21,22].
2.3. Elemental analysis using ICP-OES
The ash (2.44 g) was dissolved in high purity concen-
trated nitric acid (50 mL) and heated. This solution was
divided into 10 mL aliquots and each diluted to 100 mL.
Samples were spiked with chromium and vanadium and
standard addition was performed . For the determi-
nation of iron, a 1 mL aliquot of the diluted sample was
taken and diluted further to 100 mL using a 5% nitric
acid solution. Calibration standards were used for the
determination of iron, copper, zinc, manganese and mo-
lybdenum. The concentrations of iron, copper, zinc, chro-
mium, manganese, vanadium and molybdenum in the ash
samples were determined using ICP-OES. Each sample
was repeated three times in order to obtain standard de-
3. Results and Discussion
As a bench mark in elemental analysis, dry ashing is in-
expensive, and can be easily conducted in chemical
laboratories. Dry ashing has been under scrutiny, but one
cannot deny its simplicity and cost effective qualities.
Arguments such as possible sample loss, reaction with
the container’s surface, and incomplete ashing have been
presented [23,28]. Yet many still regard dry ashing as a
viable practice provided care is taken in the process
In the last century, attempts have been made to estab-
lish a uniform process for the pretreatment of samples
prior to metal detection. Despite efforts, only general
guidelines were formulated with specific details for vari-
ous samples. Middleton and Stuckey summarized previous
studies using either dry or wet methods for some metals
and recommended a temperature range of 500˚C - 550˚C
. Gorsuch and Thiers elaborated on this, adding more
comprehensive information for additional metals based on
ashing of numerous samples [31,32]. In order to form a
collective view of these methods, the Analytical Methods
Committee published “Methods for the Destruction of
Organic Matter” discussing wet and dry decomposition
. In this article, guidelines still in practice today were
outlined for dry ashing and advantages of the technique
over wet digestion were established. Even in more recent
works, only general guidelines are regarded and little
uniformity is seen. Tidehag and co-workers dry-ashed
blood samples for a total of 48 h but did not disclose
details or mention the total ash obtained . Dry ashing
allows for most common metals to be analyzed. The lack
of additional reagents prevents interferences in a blank
during analysis. A larger quantity of sample can be ana-
lyzed, but perhaps the most attractive trait of all is the
small amount of attention required for ash samples to be
After much experience with ashing blood, we have
developed a new procedure that uses a ceramic evapo-
rating dish and Pyrex watch glass cover rather than a
standard crucible and lid. Such a design allows for in-
creased air exposure to the sample to increase the ashing
rate. With the use of a thermocouple, the ashing tem-
perature has been closely controlled, leading to minimum
sample loss and much faster ashing time in comparison
to the control process that uses a reported crucible sys-
tem. All other variables remain consistent and are given
in detail, including initial evaporation time, ashing times,
temperatures, and overall yields (Table 1). The volume
Copyright © 2011 SciRes. AJAC
S. A. BRAGG ET AL.981
of blood to be dry-ashed depends on the size of the
evaporating dish. With our equipment, as much as 50 -
100 mL of blood could be dry-ashed in one procedure.
Closely controlling the ashing temperature has also
been found to prevent sample loss and cross reaction
with the container. Additionally, the temperature was
elevated gradually. Thus, careful monitoring of the tem-
perature is recorded in the current work in association
with the ashing time at a specific temperature. Maximum
temperature was no higher than 500˚C in order to prevent
any possible volatilization of metals as well as reaction
with the crucible container .
Another key improvement in the current procedure is
the use of a ceramic evaporating dish with a Pyrex watch
glass as a cover rather than a ceramic crucible and lid.
This modification leads to an increase in the air flow
over the sample, allowing for a significantly shorter total
ashing time. Sufficient supply of O2 in the air flow is
believed to increase the rate of the oxidization of organic
materials in the heated sample, leading to their conver-
sion to CO2 and reducing the ashing time. We have con-
ducted control studies to better demonstrate the im-
provement of this system in comparison to the literature
procedure using a crucible (and lid) and Bunsen burner.
Two experiments were performed using the same burner
and the same mass of whole blood, except that one used
a ceramic evaporating dish with a Pyrex watch glass
(Method A), and another used a crucible and lid as con-
trol (Method B). To the dish and the crucible, whole
blood (40.1 g) was added to each. Each apparatus was set
on the hot plate and allowed to evaporate water at 80˚C
for 8 h. Initial heating on the burner was allowed. The
sample in the crucible, however, required both an extra
hour of low heat and close monitoring, as its sample un-
der the lid, spilled over from the inside. After 30 min of
low heat (250˚C - 300˚C), the evaporating dish/watch
glass system was increased to 450˚C. With intermittent
stirring the total ashing time was 18 h for the new pro-
cedure (Method A). Under the same conditions the cru-
cible system in the control (Method B) required a total of
55 h of ashing. The control using the reported procedure
(Method B) leads to at least 11% loss of the ash product.
A comparison of both methods is summarized in Table
In addition to avoiding sample loss, the new procedure
(Method A) allows for the sample to be completely ashed
in less than one-third of the time used in the control
(Method B). It also involves less work. We have also
used the new method to ash, in one procedure, more
blood samples than the reported procedure using a cruci-
ble [4,23]. This is a very attractive feature, especially
when examining trace metals. If a smaller amount of
blood is needed to be ashed in, e.g., the analysis of abun-
Table 1. Ashing times and yields of evaporating dish and
period (h)Initial heating
period (h) Ashing
Method A8 0.5 18 0.4123
(Control) 8 1.5 55 0.3705
Table 2. Analysis of metals in dry-ashed whole blood.
Fe 298.1 ± 5.8 ppm
Zn 272 ± 14 ppb
Cu 169.4 ± 1.7 ppb
Cr 7.71 ± 1.7 ppb
Mn 858 ± 16 ppt
V below limit of detection
Mo below limit of detection
dant metals, the new procedure using an evaporating dish
can be completed in shorter time than 18 h.
To demonstrate both abundant and trace analysis, the
concentrations of iron, copper, zinc, chromium, manga-
nese, vanadium and molybdenum were determined from
the same blood sample using ICP-OES. Results are
shown in Table 2.
Dry ashing is a valid pretreatment technique that has
been used as a benchmark for other methods . The
current work reports a new dry ashing procedure that can
be implemented in any lab setting. Many metals may be
detected at once or individually. The method is simple,
inexpensive, and faster than the established procedure.
The dry ashing of blood samples using the new proce-
dure may help set a precedent for dry ashing of other
The authors thank the US National Institutes of Health
(1R01DK078652-01A2) for financial support and Wam-
pler’s Sausage Farm, Lenoir City, Tennessee, for pro-
viding the porcine blood samples.
 D. T. Burns, “Precursors to and Evolution of Elemental
Organic Tube Combustion Analysis over the Last Two
Copyright © 2011 SciRes. AJAC
S. A. BRAGG ET AL.
Hundred Years,” Analytical Procedures, Vol. 30, No. 6,
1993, pp. 272-275. doi:10.1039/ap9933000272
 J. Cholak and D. M. Hubbard, “Spectrographic Determi-
nation of Beryllium in Biological Material and in Air,”
Analytical Chemistry, Vol. 20, No. 1, 1948, pp. 73-76.
 J. Sysalova and V. Spevackova, “A Study of Sample Min-
eralization Methods for Arsenic Analysis of Blood and
Urine by Hydride Generation and Graphite Furnace
Atomic Absorption Spectrometry,” Central European
Journal of Chemistry, Vol. 1, No. 2, 2003, pp. 108-120.
 J. Versieck and L. Vanballenberghe, “Determination of
Tin in Human Blood Serum by Radiochemical Neutron
Activetion Analysis,” Analytical Chemistry, Vol. 63, No.
11, 1991, pp. 1143-1146. doi:10.1021/ac00011a016
 G. Cobo, M. Gomez, C. Camara and M. A. Palacios,
“Determination of Fluoride in Complex Liquid Matrices
by Electrothermal Atomic Absorption Spectrometry with
in-Furnace Oxygen-Assisted Ashing,” Microchimica Acta,
Vol. 110, No. 1-3, 1993, pp. 103-110.
 L. Vesterberg and T. Bergstrom, “Determination of Cad-
mium in Blood by Use of Atomic Absorption Spectros-
copy with Crucibles―and a Rational Procedure for Dry-
Ashing,” Clinical Chemistry, Vol. 23, 1977, pp. 555-559.
 G. Nise and O. Vesterberg, “Blood Lead Determination by
Flameless Atomic Absorption Spectroscopy,” Clinica
Chimica Acta, Vol. 84, No. 1-2, 1978, pp. 129-136.
 C. J. Price, P. L. Strong, F. J. Murray and M. M. Goldberg,
“Blood Boron Concentrations in Pregnant Rats Fed Boric
Acid Throughout Gestation,” Reproductive Toxicology,
Vol. 11, No. 6, 1997, pp. 833-842.
 K. Bukhave, A. Sørensen and M. Hansen, “A Simplified
Method for Determination of Radioactive Iron in Whole-
Blood Samples,” Journal of Trace Elements in Medicine
and Biology, Vol. 15, No. 1, 2001, pp. 56-58.
 W. Jiang, H. Tong, Z.-C. Liu, K. Wang, B.-C. Chen and
C.-H. Li, “Determination of Whole Blood Lead by Dry
Ashing-GFAAS,” Chinese Journal of Health Laboratory
Technology (Zhongguo Weisheng Jianyan Zazhi), Vol. 18,
No. 3, 2008, pp. 464-465.
 L. Cao and Y. He, “Determination of Blood Lead and
Cadmium Levels by Dry-Ash Graphite Furnace Atomic
Absorption Spectrometry,” Occupation and Health (Zhiye
Yu Jiankang), Vol. 24, No. 23, 2008, pp. 2536-2537.
 J. Titze, H. Krause, H. Hecht, P. Dietsch, J. Rittweger, R.
Lang, K. A. Kirsch and K. F. Hilgers, “Reduced Osmoti-
cally Inactive Na Storage Capacity and Hypertension in
the Dahl Model,” American Journal of Physiology―Re-
nal Physiology, Vol. 283, No. 1, 2002, pp. F134-F141.
 K. Drábek and J. Kalousková, “Comparison of Recover-
ies of Inorganic and Organic Incorporated 75-Se by Four
Mineralization Methods of Biological Material,” Journal
Radioanalytical and Nuclear Chemistry, Vol. 119, No. 2,
1987, pp. 119-129. doi:10.1007/BF02169840
 J. Titze, J. Rittweger, P. Dietsch, H. Krause, K. H.
Schwind, K. Engelke, R. Lang, K. A. Kirsch, F. C. Luft
and K. F. Hilgers, “Hypertension, Sodium Retention, Cal-
cium Excretion and Osteopenia in Dahl Rats,” Journal of
Hypertension, Vol. 22, 2004, pp. 803-810.
 J. Bian, X. Zhang and W. Ni, “Determination of Alumi-
num in Flour Products by Microwave Digestion/Dry
Ashing-Spectrophotometry,” Chemistry Research
(Huaxue Yanjiu), Vol. 22, No. 2, 2011, pp. 61-64.
 V. M. Tomovic, L. S. Petrovic, M. S. Tomovic, Z. S.
Kevresan, M. R. Jokanovic, N. R. Dzinic and A. R.
Despotovic, “Cadmium Levels of Kidney from 10 Dif-
ferent Pig Genetic Lines in Vojvodina (Northern Ser-
bia),” Food Chemistry, Vol. 129, No. 1, 2011, pp. 100-
 C. R. Brown, K. G. Haynes, M. Moore, M. J. Pavek, D. C.
Hane, S. L. Love, R. G. Novy, J. C. Miller Jr., “Stability
and Broad-Sense Heritability of Mineral Content in Po-
tato: Zinc,” American Journal of Potato Research, Vol.
88, No. 3, 2011, pp. 238-244.
 N. Sogabe, R. Maruyama, O. Baba, T. Hosoi and M. Go-
seki-Sone, “Effects of Long-Term Vitamin K1 (Phyllo-
quinone) or Vitamin K2 (Menaquinone-4) Supplementa-
tion on Body Composition and Serum Parameters in
Rats,” Bone, Vol. 48, No. 5, 2011, pp. 1036-1042.
 A. L. R. M. Rossete, J. M. T. Carneiro, H. H. Batagello, J.
G. G. Oliveira and J. A. Bendassolli, “Spectrophotometric
Determination of Sulfur in Plants Using Dry Ash Oxida-
tion and Alkaline Oxidizers,” Quimica Nova, Vol. 34, No.
2, 2011, pp. 341-343.
 J. Vogl, M. Rosner and W. Pritzkow, “Development and
Validation of a Single Collector SF-ICPMS Procedure for
the Determination of Boron Isotope Ratios in Water and
Food Samples,” Journal of Analytical Atomic Spectrome-
try, Vol. 26, No. 4, 2011, pp. 861-869.
 L. Yong, K. C. Armstrong, R. N. Dansby-Sparks, N. A.
Carrington, J. Q. Chambers and Z. Xue, “Quantitative
Analysis of Trace Chromium in Blood Samples. Combi-
nation of the Advanced Oxidation Process with Catalytic
Adsorptive Stripping Voltammetry,” Analytical Chemistry,
Vol. 78, No. 21, 2006, pp. 7582-7587.
 R. N. Dansby- Sparks, R.-Z. Ouyang and Z.-L. Xue,
“Optical and Electro-Chemical Sol-Gel Sensors for Inor-
ganic Species,” Science in China Series B: Chemistry,
Vol. 52, No. 11, 2009, pp. 1777-1788.
 R. Bock, “A Handbook of Decomposition Methods in
Analytical Chemistry,” International Textbook Company,
 Analytical Methods Committee, “Methods for the De-
Copyright © 2011 SciRes. AJAC
S. A. BRAGG ET AL.
Copyright © 2011 SciRes. AJAC
struction of Organic Matter,” Analyst, Vol. 85, 1960, pp.
 P. F. E. Van Montfort, J. Agterdenbos and B. A. H. G.
Juette, “Determination of Antimony and Tellurium in Hu-
man Blood by Microwave Induced Emission Spectrome-
try,” Analytical Chemistry, Vol. 51, No. 9, 1979, pp.
 A. Ferrando, N. Green, K. Barnes and B. Woodward,
“Microwave Digestion Preparation and ICP Determina-
tion of Boron in Human Plasma,” Biological Trace Ele-
ment Research, Vol. 37, 1993, pp. 17-25.
 D. C. Harris, “Quantitative Chemical Analysis,” Mac-
 P. Mader, J. Száková and E. Curdová, “Combination of
Classical Dry Ashing with Stripping Voltammetry in
Trace Element Analysis of Biological Materials: Review
of Literature Published after 1978,” Talanta, Vol. 43, No.
4, 1996, pp. 521-534. doi:10.1016/0039-9140(95)01793-3
 P. Tidehag, G. Hallmans, K. Wing, R. Sjöström, G. ÅGren,
E. Lundin and J. Zhang, “A Comparison of Iron Absorp-
tion from Single Meals and Daily Diets Using Radio Fe
(Fe, 59Fe),” British Journal of Nutrition, Vol. 75, 1996, pp.
 G. Middleton and R. E. Stuckey, “The Preparation of Bio-
logical Material for the Determination of Trace Metals.
Part I. A Critical Review of Existing Procedures,” Analyst,
Vol. 78, 1953, pp. 532-542. doi:10.1039/an9537800532
 T. T. Gorsuch, “Radiochemical Investigations on the Re-
covery for Analysis of Trace Elements in Organic and
Biological Materials, Report to the Analytical Methods
Committee by the Society’s First Analytical Chemistry
Research Scholar,” Analyst, Vol. 84, 1959, pp.135-173.
 R. E. Thiers, “Contamination in Trace Analysis and Its
Control,” Methods of Biochemical Analysis, Vol. 5, 1957,
pp. 273-335. doi:10.1002/9780470110218.ch6