Optimization of Dry Ashing of Whole Blood Samples for Trace Metal Analysis

doi:10.4236/ajac.2011.28114

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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)

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

E-mail: [email protected]

Received July 20, 2011; revised September 14, 2011; accepted October 3, 2011

Abstract

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

1. Introduction

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

reported here.

2. Experimental

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.

980

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) [23]

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 [27]. 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-

viations.

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

[9-12,14,21,22,29].

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

[30]. 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

[24]. 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 [29]. 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

completed [24].

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

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 [30].

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

crucible.

Evaporation

period (h)Initial heating

period (h) Ashing

period (h)Ash

mass (g)

Method A8 0.5 18 0.4123

Method B

(Control) 8 1.5 55 0.3705

Table 2. Analysis of metals in dry-ashed whole blood.

Element Concentration

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.

4. Conclusions

Dry ashing is a valid pretreatment technique that has

been used as a benchmark for other methods [4]. 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

samples.

5. Acknowledgements

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.

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