Practicality of Tetragonal Nano-Zirconiaas a Prospective Sorbent in the Preparationof 99Mo/99mTc Generator for BiomedicalApplications

Rubel Chakravarty1, Rakesh Shukla2, Ramu Ram1, Avesh Kumar Tyagi2, Ashutosh Dash1,&,Meera Venkatesh1

1 Radiopharmaceuticals Division, Bhabha Atomic Research Centre, Mumbai, India; E-Mail: adash@barc.gov.in2 Chemistry Division, Bhabha Atomic Research Centre, Mumbai, India

Received: 18 May 2010 / Revised: 3 August 2010 / Accepted: 20 August 2010Online publication: 26 September 2010

Abstract

The feasibility of using tetragonal nano-zirconia (t-ZrO2) as an effective sorbent for devel-oping a 99Mo/99mTc chromatographic generator was demonstrated. The structural charac-teristics of the sorbent matrix were investigated by different analytical techniques such asXRD, BET surface area analysis, FT-IR, TEM etc. The material synthesized was nanocrystal-line, in tetragonal phase with an average particle size of *7 nm and a large surface area of340 m2 g-1. The equilibrium sorption capacity of t-ZrO2 is >250 mg Mo g-1. The presentstudy indicates that 99Mo is both strongly and selectively retained by t-ZrO2 at acidic pH and99mTc could be readily eluted from it, using 0.9% NaCl solution. A 9.25 GBq (250 mCi)t-ZrO2 based chromatographic 99Mo/99mTc generator was developed and its performancewas repeatedly evaluated for 10 days. 99mTc could be eluted with >85% yield havingacceptable radionuclidic, radiochemical and chemical purity for clinical applications.The compatibility of the product in the preparation of 99mTc labeled formulations such as99mTc-EC and 99mTc-DMSA was evaluated and found to be satisfactory.

Keywords

Chromatographic 99Mo/99mTc generatorSorption capacityDistribution ratioZeta potentialNano-zirconia

Introduction

The vital role of 99mTc in the field of

diagnostic nuclear medicine is well

established. It is used for approximately

20–25 million procedures annually,

comprising *80% of all diagnostic nu-

clear medicine procedures [1, 2]. Over the

past 50 years, several versions [3–10] of

the 99Mo/99mTc generator have been

developed, the most common being the

original alumina based chromatographic

generator system. The ease of operation,

high elution efficiency, high radionucli-

dic purity and radioactive concentration

of 99mTc eluate are attractive features of

these generator systems. The capacity of

alumina for taking up molybdate ions is

limited (2–20 mg Mo g-1 of alumina) [4]

necessitating the use of 99Mo of the

highest specific activity available, gener-

ally possible only in 99Mo produced

through fission route. The separation of

fission 99Mo, involves an elaborate and

complex processing technology which is

expensive, generates huge quantities of

radioactive wastes and requires extensive

purification prior to use.

2010, 72, 875–884

DOI: 10.1365/s10337-010-1754-z0009-5893/10/11 � 2010 Vieweg+Teubner Verlag | Springer Fachmedien Wiesbaden GmbH

Original Chromatographia 2010, 72, November (No. 9/10) 875

In order to minimize the dependence

on the fission produced 99Mo, several

alternative approaches like ‘batch’ sol-

vent extraction [7] and dry distillation of99mTc from (n, c)99Mo[MoO3] target [5]

were reported for accessing 99mTc from

(n, c)99Mo. The need for strict adherence

to the operational protocol, good labo-

ratory practices from both pharmaceuti-

cal and radiological safety angles and the

requirement for well-trained, skilled

operator are the major roadblocks in the

path of the success of these techniques.

The need for a simple user-friendly col-

umn based generator that uses (n,

c)99Mo, prompted researchers in explor-

ing the gel generator route [8–10]. The

developments over the last two decades

however have shown that it has inherent

disadvantages which limit its wide

applicability. Attempts to develop sys-

tems with a primary alumina column

generator containing (n, c)99Mo, in tan-

dem with a device to concentrate a large

volume of eluate in an acceptable small

volume of <5 mL [11–14] have also been

tried with limited success. There has also

been widespread interest in the develop-

ment of 99Mo/99mTc generators from (n,

c)99Mo using alternate sorbents having

higher sorption capacity compared to

alumina [15–17]. Masakazu et al. [17]

developed a 99Mo/99mTc generator using

polymeric zirconium compound (PZC)

which showed high sorption capacity for99Mo (*200 mg g-1). However, the slow

kinetics of sorption and appreciably high99Mo breakthrough [17] are the signifi-

cant drawbacks that limit its applicability

in a clinical context.

The conventional inorganic sorbents

have low sorption capacity due to the

limited active sites and relatively low

accessible surface area. Hence, such

sorbents cannot be used for the prepa-

ration of 99Mo/99mTc generators utiliz-

ing low specific activity (n, c)99Mo. One

of the effective approaches to increase

their accessible surface area and active

sites is to nanoengineer the bulk parti-

cles. The surface atoms of nanoparticu-

late metal oxides are unsaturated, exhibit

intrinsic surface reactivity and can be

used in the chromatographic separation

of metal ions [18–21]. In order to tap the

potential of nanomaterials as effective

sorbents in the preparation of radionu-

clide generators, our group has success-

fully demonstrated the development of99Mo/99mTc [22], 188W/188Re [23, 24] and68Ge/68Ga generators [25] using this class

of compounds.

In the current study, we aimed at

developing a 99Mo/99mTc generator

using tetragonal nano-zirconia (t-ZrO2)

as a new generation sorbent. t-ZrO2 was

selected for this purpose mainly because

of the effectiveness of hydrous zirconia

as an ion-exchanger for the separation

of metal ions [19, 20]. Though a large

number of techniques for the synthesis

of high surface area nano-zirconia have

been reported [26–33], the simple, cost-

effective direct precipitation method of

Yin et al. [32], appeared to be the most

attractive and therefore adapted in our

investigation. In this communication,

we describe the effectiveness of t-ZrO2

in the development of 99Mo/99mTc gen-

erator. The feasibility of the method,

both in terms of yield and the purity of

the 99mTc for radiopharmaceutical

application, has been demonstrated and

evaluated.

Experimental

Chemicals

Reagents such as hydrochloric acid,

ammonium hydroxide, etc. were of ana-

lytical grade and were procured from

S. D. Fine Chemicals, Mumbai, India.

ZrOCl2 � 8H2O (99.9%) was from

E. Merck, Darmstadt, Germany. Dim-

ercaptosuccinic acid (DMSA) and eth-

ylene dicysteine (EC) kits were from

the Board of Radiation and Isotope

Technology, Navi Mumbai, India.

Paper chromatography strips were pur-

chased from Whatman, Maidstone, UK.

Molybdenum-99 (specific activity

11.1–18.5 GBq g-1 of 300–500 mCi g-1)

as Sodium (99Mo) Molybdate was

available in the Radiopharmaceuticals

Division of our Institute. For the deter-

mination of the distribution ratio of99Mo in t-ZrO2, lower specific activity99Mo (*11.1 GBq g-1) was used while

for the preparation of the 99Mo/99mTc

generator, freshly produced 99Mo of

higher specific activity (*18.5 GBq g-1)

was used.

Instrumentation

Gamma activity of 99mTc was assayed

using a NaI (Tl) scintillation counter

(100–160 keV). HPGe detector coupled

with a multichannel analyzer (MCA)

(Canberra Eurisys, France) with a

1.5 keV resolution at 1,333 keV ranging

from 1.8 to 2 MeV was used for the

analysis of 99Mo in the presence of 99mTc

and also for quantitative estimation. The

radioisotope levels were determined by

quantification of the photo peaks corre-

sponding to 99mTc [140 keV (88.97%)]

and 99Mo [181 keV (6%) and 740 keV

(12.2%)]. The zeta-potential of the

nanoparticles were measured using a

Zetasizer (Nano ZS/ZEN3600, Malvern

Instruments, UK). The chemical analysis

for the determination of trace level of

metal contaminations was done using

inductively coupled plasma-atomic

emission spectroscopy (ICP-ES JY-238,

Emission Horiba Group, France). X-ray

diffraction measurements (10–70�) were

carried on the powder for the crystallite

size estimation, using monochromatized

Cu–Ka radiation on a Philips X-ray

diffractometer, Model PW 1927. Silicon

was used as an external standard for

correction due to instrumental broaden-

ing. FT-IR spectra of the synthesized

sorbent were recorded in a JASCO FT–

IR-420 spectrometer. Thermogravimetry

was carried out using Setaram Simulta-

neous TG-DTA (Model 92-16.18)

instrument. The analysis was conducted

in argon atmosphere with the flow rate

60 mL min-1 at a heating rate of 10 �Cmin-1, using a-alumina as the reference

in a platinum crucible. TEM data were

recorded using TEM, JEOL FX micro-

scope (Jeol, Tokyo, Japan) on the pow-

der sample. The preparation of samples

for TEM analysis involved sonication in

ethanol for 5 min and deposition on a

carbon coated copper grid. The acceler-

ating voltage of the electron beam was

200 kV.

Synthesis and StructuralCharacterization of t-ZrO2

High surface area t-ZrO2 was synthe-

sized by controlled hydrolysis of zirconyl

chloride in ammonical medium as per

876 Chromatographia 2010, 72, November (No. 9/10) Original

the procedure reported by Yin et al. [32].

0.17 M zirconyl chloride was added

drop-wise into a round bottom flask

containing 2.5 M ammonia solution,

with careful control of pH (9–11) and

with vigorous stirring. The resultant

hydrogel was washed with de-ionized

water until free of chloride ions. The

hydrogel was then digested under reflux

at 96 �C for 24 h in a 1 L round bottom

flask containing aqueous solution of

ammonia (pH * 12). Subsequently, the

digested gels were washed extensively

with de-ionized water. The washed gel

was dried at 100 �C overnight. The dried

gel was calcined at 600 �C for 5 h and

used for structural studies. Further, it

was ground in a porcelain mortar and

sieved to get particles of 50–100 mesh

(149–297 lm). The structural character-

ization of t-ZrO2 was carried out by

various analytical techniques such as

XRD, IR spectroscopy, BET surface

area and pore size analysis, thermal

analysis, TEM etc.

The chemical stability of t-ZrO2 was

assessed in several mineral acids and

bases, such as HCl, HNO3, NaOH and

NH4OH as per the reported procedure

[23, 24].

Determination of theDistribution Ratio (Kd)

Distribution ratios (Kd) of MoO42- and

TcO4- ions for the t-ZrO2 matrix were

measured at different pH, using 99Mo

and 99mTc radiotracers. For estimation

of Kd values for 99Mo, 99Mo/99mTc

mixture was used, while for 99mTc, pure99mTc was used. In each experiment,

200 mg of sorbent was suspended in

20 mL solution containing the radioac-

tive metal ions, in a 50 mL stoppered

conical flask. The flasks were shaken in

a wrist arm mechanical shaker for

30 min at 25 �C and then filtered. The

activities of the solution before and

after equilibration were measured in a

HPGe counter using an appropriate

c-ray peak (140 keV for 99mTc and

181 keV for 99Mo). The distribution

ratios were calculated using the follow-

ing expression:

Kd ¼ðAi � AeqÞ V

Aeq mLg�1

where Ai is the initial total radioactivity

of 1 mL the solution, Aeq is the unad-

sorbed activity in 1 mL of the solution at

equilibrium, V is the solution volume

(cm3) and m is the mass (g) of the

sorbent.

Determination of the Time ofEquilibration

In order to study the time dependence of

sorption of 99Mo onto t-ZrO2, distribu-

tion ratio (Kd) of 99MoO42- ions in

0.001 M HNO3 solution was determined

at different time intervals (5–40 min), as

described above. The Kd values (mean of

three experiments) were taken as an

indication of the progress of the sorption

process. The time when Kd remained

unchanged, was taken as the indication

of the attainment of equilibrium.

Determination of ZetaPotential

Approximately, 5 mg of the sorbent

were added to 50 mL of de-ionized water

and the pH of the suspension was ad-

justed using HClO4 and NH4OH. Zeta

potential of the suspensions at different

pH was measured with a combination of

laser Doppler velocimetry and phase

analysis light scattering (PALS). Smolu-

chowsky constant F(Ka) of 1.5 was used

to calculate the zeta potential values

from the electrophoretic mobility. All

measurements were carried out at 25 �Cin triplicate.

Determination of SorptionCapacity of t-ZrO2

Static Sorption Capacity

The ion exchange capacity of the sorbent

for Mo was determined by batch equili-

bration method. 0.5 g of accurately

weighed sorbent were taken in a stop-

pered conical flask and equilibrated with

50 mL of sodium molybdate solution

(10 mg of Mo mL-1) spiked with

*370 kBq (10 lCi) of 99Mo for 30 min

at room temperature. At the end, the

contents were filtered through a What-

man filter paper (No. 542). The activities

of 99Mo in the solution before and after

absorption were estimated by using a

HPGe detector coupled to a multichan-

nel analyzer, by measuring the counts at

181 keV peak corresponding to 99Mo in

1 mL aliquots. All measurements were

carried out at 25 �C in triplicate. The

capacity was calculated using the fol-

lowing expression:

Capacity ¼ ðC0 � CeÞ Vm

where, C0 and Ce represented initial and

equilibration concentrations of Mo

respectively, V was the volume of solu-

tion and m was the mass (g) of the

sorbent.

Dynamic Sorption Capacity

In order to estimate the Mo sorption

capacity under dynamic conditions, a

borosilicate glass column 15 cm 9

0.4 cm (i.d.) with sintered disc (G0) at the

bottom was packed with 0.5 g of the

synthesized sorbent. After the column

was conditioned with 0.001 M HNO3,

sodium molybdate solution (10 mg

Mo mL-1), spiked with 99Mo tracer

[370 kBq (10 lCi)] was allowed to pass

through the column at a rate of

0.25 mL min-1. 1 mL of this solution

was kept as reference (C0). The effluent

was collected in fractions of 1 mL ali-

quots (C). The 99Mo activity in the ref-

erence (C0) and effluent fractions were

determined by measuring the 181 keV c-ray peak of 99Mo in a HPGe detector.

The ratio of the count rate ‘C’ of each

1 mL effluent to the count rate ‘C0’ of

1 mL of the original feed Mo solution

was taken as the parameter to follow the

sorption pattern.

Practical Sorption Capacity

In order to determine the maximum

achievable sorption capacity under col-

umn conditions, a borosilicate glass col-

umn of dimension 30 cm 9 0.4 cm (i.d.)

Original Chromatographia 2010, 72, November (No. 9/10) 877

with sintered disc (G0) the bottom was

packed with 1 g of the synthesized sor-

bent. After the column was conditioned

with 0.001 M HNO3, 20 mL of sodium

molybdate solution (10 mg Mo mL-1,

pH 3), spiked with 99Mo tracer (370 kBq)

was added into the column and allowed

to stand under static conditions for 1 h.

1 mL of this solution was kept as refer-

ence. Subsequently, the solution was al-

lowed to pass through the column at a

flow rate of 0.25 mL min-1. The column

was washed with 50 mL of 0.9% NaCl

solution. The effluents were pooled to-

gether and the activity was determined.

The sorption capacity was determined by

comparing the 99Mo activity in the efflu-

ent with that added into the column.

Development of 99Mo/99mTcGenerator

A borosilicate glass column of dimen-

sion 30 cm 9 0.4 cm (i.d.) with sintered

disc (G0) at the bottom was packed with

4 g of t-ZrO2 in a lead shield. It was

preconditioned with 0.001 N nitric acid

solution at pH 3. The column was then

loaded by adding 10 mL of Na2[99-

MoO4] (9.25 GBq of 99Mo, specific

activity 17.8 GBq g-1) solution main-

tained at pH 3, into the column and

allowing it to stand under static condi-

tions for 1 h. Subsequently, the 99Mo

solution was allowed to pass through the

column at a flow rate of 0.25 mL min-1,

by applying suction using a peristaltic

pump. The column was then washed

with 100 mL of 0.9% NaCl solution.

Elution of 99mTc from theGenerator

In order to study the kinetics of

desorption of 99mTc from the t-ZrO2

column, it was eluted with 0.9% NaCl

solution maintaining different flow rates

(0.25–2 mL min-1), using a peristaltic

pump. The activity of 99mTc was mea-

sured in an ionization chamber and the

elution yield was determined. Further, in

order to study the elution profile of the

generator, it was eluted with 0.9% NaCl

solution at a flow rate of 1 mL min-1.

The eluate was collected in a series of

1 mL aliquots, and the activity of each

sample was measured. All runs were

performed at room temperature. The

generator was regularly eluted with

4 mL of 0.9% NaCl solution for

10 days.

Quality Control of 99mTc Eluate

Radionuclidic Purity

Radionuclidic purity measurements were

made using a calibrated HPGe detector.

The 99Mo contamination level in 99mTc

was quantified by allowing the 99mTc

samples to decay for 2 days and then

measuring the 181 keV c-ray peak, cor-

responding to emission from 99Mo con-

taminant. Further, the radionuclidic

purity of 99mTc was evaluated by the

determination of its half-life.

Radiochemical Purity

To evaluate the radiochemical purity of99mTcO4

-, 5 lL of activity was applied

on paper chromatographic strips

(Whatman 12 cm 9 1 cm) at 1.5 cm

from the lower end. The strips were

developed in 85% methanol solution.

After chromatography, the paper strip

was dried, cut into 1 cm segments,

placed in test tubes and counted in a

NaI(Tl) scintillation counter.

Chemical Purity

In order to determine the presence of

metal contamination (chemical impuri-

ties), the 99mTc samples were allowed to

decay for 10 days. The presence of trace

level of Zr ion contamination in the de-

cayed samples was determined by

inductively coupled plasma-atomic

emission spectroscopy (ICP-AES).

Labeling Efficacy

99mTc eluted from the generator was

used to prepare complexes of dim-

ercaptosuccinic acid (DMSA) and eth-

ylene dicysteine (EC) using standard

commercial kits, provided by the Board

of Radiation and Isotope Technology,

India.

Recovery of Mo fromthe Spent Generator andRegeneration of the Column

The spent generator was washed with

saline, and the Mo was desorbed with

3 M NaOH solution containing H2O2

(15 mL of 5 M NaOH solution + 1 mL

of 30% H2O2). The flow rate of the

eluent was *1 mL min-1. The eluate

was collected in a series of 1 mL aliquots

throughout the elution, and each sample

was then subjected to c-ray spectrome-

try. All the fractions were mixed together

and the total activity of 99Mo eluted was

determined. The column was further

washed with de-ionized water and then

conditioned with nitric acid solution at

pH 3. 99Mo activity was re-loaded in

the column and 99mTc was eluted out

under the same conditions as mentioned

earlier.

Results

Synthesis and StructuralCharacterization of t-ZrO2

It was essential to add zirconium oxy-

chloride solution to the NaOH solution

in the reaction vessel, with vigorous

stirring. This order of addition is

important as formation of zirconia pre-

cipitate in alkaline environment helps in

stabilization of the precursor to t-ZrO2

nanoparticles [28, 33]. The vigorous

stirring of the reaction mixture prevents

agglomeration of the precipitate and

hence facilitates the formation of the

nanoparticles. The reflux digestion of the

basic solution in the glass vessel leads to

stabilization of small-size tetragonal

crystals at high temperature. After

establishing appropriate synthesis pro-

cedure, several batches of t-ZrO2 sorbent

were prepared. The existing synthesis

protocol is capable of delivering

about 5 g of material per batch. The

product obtained in all the batches was

granular with adequate mechanical

strength amenable for fixed-bed column

operation.

The XRD pattern of t-ZrO2 is shown

in Fig. 1. The results suggest that the

obtained product has high degree of

878 Chromatographia 2010, 72, November (No. 9/10) Original

crystallinity. The relatively broad peaks

are attributed to the very small crystallite

size of the material. The XRD studies

showed that the major constituent phase

is tetragonal. The average crystallite size

of t-ZrO2 as determined from the XRD

pattern using Debye–Scherrer’s method

[24–26] was *7 nm. The infrared sor-

bent spectrum of synthesized adsorbent

is shown in Fig. 2. The sample as such

showed a broad absorption peak in the

range 3,600–3,000 cm-1, which is due to

the sum of the contributions of hydroxyl

groups and water molecules. Absorption

peak at 1,614 cm-1 was due to the

bending mode of OH- group attached to

the matrix. The bands at 1,000 cm-1,

490–501 cm-1 are attributed to Zr–O–Zr

bond. The surface area measurements

and the pore size distribution of the

sorbents were carried out by standard

BET technique. The surface area of

t-ZrO2 was found to be *340 m2 g-1.

The average pore size was determined to

be *4 A. It was observed that pores

sizes are uniform which facilitate per-

mutation of liquid. The TEM micro-

graph (Fig. 3) indicated that the t-ZrO2

was highly agglomerated and the parti-

cles are quite uniform in size and shape.

The average crystallite size (of primary

particles) of t-ZrO2 as determined by the

TEM measurements was found to be in

the range of 6–10 nm, which is in

accordance with the results obtained

from the XRD method. These nano-

sized crystallites have high surface en-

ergy. It is an established observation that

in order to stabilize themselves, the

nano-sized crystallites get agglomerated

to form micron sized granules (com-

prising a large number of primary par-

ticles). Such granules were crushed and

sieved to get particles of 50–100 mesh

size. The agglomeration of the crystal-

lites and the subsequent crushing and

sieving of the agglomerates renders free

flowing granular particles of t-ZrO2

suitable for use in column operations.

The TG curve of the sorbent is depicted

in Fig. 4. It can be seen from the curve

that at a temperature of *100–180 �C,the material shows a loss of *14% of its

initial weight. This weight loss can be

attributed to loss of physically adsorbed

water. After the loss of water, there is no

significant weight change up to a tem-

perature of 1,000 �C. From the data, it

can be inferred that the sorbent material

is very well stable up to a temperature of

1,000 �C.It was observed that t-ZrO2 was

insoluble in water, dilute mineral acids

and alkali (up to 6 M) as <0.1 ppm le-

vel of Zr ions were detected in the filtrate

when analyzed by ICP-AES. It is clear

that an appreciable amount of dissolu-

tion did not take place and the sorbent

was fairly stable in most of the dilute

mineral acids and alkalis. There was no

turbidity when the sorbent was sus-

pended in water for 24 h. This charac-

teristic shows that the sorbent can safely

be used for generator preparation.

Determination of theDistribution Ratio (Kd)

In order to verify the usefulness of the

synthesized material for the sorption of99Mo from aqueous solutions and selec-

tive elution of 99mTc, distribution ratio

(Kd) were determined. The distribution

ratio (Kd) results for99Mo and 99mTc in

t-ZrO2 are summarized in Table 1. It is

apparent that the Kd values for Mo and

Tc attained the maximum value at

around pH 3 and hence this medium is

suitable for sorption of 99Mo. In 0.9%

NaCl solution, the high Kd values for99Mo clearly indicate that it can be effi-

ciently retained by the sorbent, while the

low Kd values for 99mTc indicate that it

shall not be retained and hence eluted

out easily.

Determination of the Time ofEquilibration

The time duration required for the

complete sorption of the 99Mo onto

t-ZrO2 was studied by following the

distribution ratio (Kd) at different time

intervals which was an indication of the

progress of the adsorption process.

It was observed that the Kd values

remained almost constant (*600) after

all the time intervals. This indicates that

the equilibrium was reached within

5 min.

Determination of ZetaPotential

In order to get a better insight about the

adsorption behavior of polymolybdate

10 20 30 40 50 60 70

t-ZrO27nm

Inte

nsit

y (a

.u)

2θ (degrees)

Fig. 1. XRD pattern of t-ZrO2

4000 3500 3000 2500 2000 1500 1000 500 0

Wavenumber (cm-1)

%T

(a.

u.)

Fig. 2. FT-IR spectra of t-ZrO2

Fig. 3. TEM micrograph of t-ZrO2

200 400 600 800 1000-16

-14

-12

-10

-8

-6

-4

-2

0

2TG - Curve

Cha

nge

in w

t. (%

)

Temperature ( oC)

Fig. 4. Thermogravimetric analysis of t-ZrO2

Original Chromatographia 2010, 72, November (No. 9/10) 879

anions on the t-ZrO2 sorbent, it was felt

necessary to determine the zeta potential

of the adsorbents at various pH values.

The effect of pH on zeta potential of

t-ZrO2 in aqueous solution is shown in

Fig. 5. It is clearly evident from the figure

that interaction of t-ZrO2 particles with

aqueous solutions imparts a pH-depen-

dent surface charge. The zeta potential

value is positive in the pH range 1–4. On

further increase of pH, the zeta potential

value passes through zero (iso-electric

point) and then it develops negative zeta

potential. The point of zero charge,

pHpzc, determined from the figure occurs

at pH 4.5 ± 0.1. From the result, it was

observed that the pH value of the 99Mo

feed solution is supposed to be less than

the pHpzc (pH value of *4.5), which

leads to the surface possessing positive

charges, so as to facilitate the sorption of

anionic polymolybdate species. This

observation is almost in agreement with

the Kd studies.

Determination of SorptionCapacity of t-ZrO2

Static Sorption Capacity

The value of the static sorption capacity

for t-ZrO2 is (250 ± 10) mg Mo g-1 of

sorbent (n = 10). This result clearly

illustrates the superiority of this material

over alumina with respect to sorption

capacity, whose adsorption capacity is

only 2–20 mg Mo g-1 of alumina [4].

Dynamic Sorption Capacity

Under dynamic condition the distribu-

tion of loaded Mo as followed through

the activity measurement is depicted in

Fig. 6. It should be noted that the

breakthrough capacity in case of t-ZrO2

is *80 mg Mo g-1, which is much

higher than that of alumina

(*2–20 mg Mo g-1 of alumina) [4].

Therefore the superiority of this material

over alumina in terms of dynamic sorp-

tion capacity has been amply demon-

strated. The sorption capacity of t-ZrO2

prepared in 10 different batches is shown

in Table 2. It is clear from the table that

the sorption capacity of the material was

consistently high in all batches.

Practical Sorption Capacity

The maximum achievable sorption

capacity under column conditions was

155 ± 12 mg Mo g-1 of sorbent

(n = 10), which is much higher than the

maximum sorption capacity of alumina

(20 mg Mo g-1) [4].

Performance of the99Mo/99mTc Generator

The performance of t-ZrO2 as a column

matrix for 99Mo/99mTc generator was

evaluated by developing a 99Mo/99mTc

generator of *9.25 GBq (250 mCi)

activity, and the elution behavior of99mTc was further studied. The elution

yield of 99mTc from the generator, at

different flow rates, is illustrated in

Fig. 7. It can be seen from the figure that

the elution yield increases on decreasing

the flow rate of the eluent. The optimum

elution yield (*85%) could be achieved

at a flow rate of 1 mL min-1, and hence

Table 1. Distribution ratios (Kd) of 99Moand 99mTc in different media

Medium (pH) Kd

99Mo 99mTc

1 129 32 295 143 611 354 560 295 525 2.76 174 0.37 34 0.18 10 0.60.9% NaCl 270 0.2

0 1 2 3 4 5 6 7 8-60

-40

-20

0

20

40

60

pHpzc

Zet

a po

tent

ial (

mV

)

pH

Fig. 5. Determination of zeta potential of t-ZrO2 as a function of pH

0 50 100 150 200 2500.0

0.2

0.4

0.6

0.8

1.0Amount of sorbent used = 500 mg

C/C

0

Amount of Mo (mg)

Fig. 6. Distribution of loaded Mo underdynamic conditions

Table 2. Sorption capacity of t-ZrO2 in 10different batches

Batch No. Staticcapacity(mg Mo g-1)

Dynamiccapacity(mg Mo g-1)

1 265 842 289 803 310 824 255 855 280 886 290 897 275 858 300 879 285 8310 298 92

0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.000

20

40

60

80

100

Elu

tion

yie

ld o

f 99

mT

c (%

)

Flow rate (mL min-1 )

Fig. 7. Elution yield of 99mTc from the gen-erator at different flow rates

0 2 4 6 8 100

20

40

60

80

100

Cum

mul

ativ

e yi

eld

of 99

mT

c (%

)

Volume of 0.9% NaCl eluent (mL)

Fig. 8. Elution profile of the generator

880 Chromatographia 2010, 72, November (No. 9/10) Original

this flow rate was maintained for all

subsequent elutions. In order to optimize

the minimum volume of eluent required

for elution of 99mTc with maximum

yield, the elution profile of the generator

was studied and is illustrated in Fig. 8. It

was observed that >80% of 99mTcO4-

available in the generator was eluted

within the first 4 mL volume of normal

saline. Thus, 99mTc could be eluted from

the generators with appreciable radio-

active concentration. Table 3 summa-

rizes the results of operation of the99Mo/99mTc generator. It was observed

that generator containing t-ZrO2 gives

consistently high yields (*90%) of99mTc over a prolonged period for

10 days.

Quality Control of 99mTc Eluate

Radiochemical Purity

To separate pertechnetate from 99mTc

present in other oxidation states, paper

chromatography (PC), developed in

85% methanol medium was used. The

paper chromatographic pattern of99mTc in 85% methanol medium is

shown in Fig. 9. The radiochemical

purity of 99mTc in pertechnetate form

was >99%, which was within the pre-

scribed limits as per the British Phar-

macopoeia [34].

Radionuclidic Purity

In order to utilize 99mTc obtained from

the generator for medical applications,

the presence of radionuclidic impurities

of the parent radioisotope must be

within the prescribed limits [34]. 99Mo

breakthrough in the 99mTc eluate was

studied by several means. The 99mTc

activity was allowed to decay for 2 days

and then its half-life was followed using

a NaI (Tl) detector. The half life of99mTc obtained was 6.1 ± 0.3 h (n = 5),

which agreed well with the 6 h half-life

of 99mTc. 99mTc eluted from the genera-

tor was checked in a HPGe detector to

see the peaks due to the c-rays corre-

sponding to 99Mo gamma activity. The

c-spectrum as illustrated in Fig. 10, did

not show the presence of such peaks,

thereby confirming the absence of 99Mo

in the eluted 99mTc. The analysis of the

decayed samples of 99mTc by c-spectro-scopic technique revealed that the level

of 99Mo present in the eluate was below

10-4% in all batches of 99mTc.

Chemical Purity

The presence of chemical impurities, in

the form of bleeding of the column ma-

trix, affects the labeling efficacy of 99mTc

and may also add an undesired amount

of chemical toxicity into the eluate. This

was analyzed by ICP-AES and the level

of Zr ions present was found to be

<0.1 ppm. It is clear that an appreciable

amount of Zr ions contamination was

not present in the 99mTc eluate.

Labeling Efficacy

In order to examine the suitability of99mTcO4

- obtained from the generators,

for radiolabeling studies, it was com-

plexed with commonly available ligands

like DMSA and EC. The paper chro-

matography patterns for 99mTc-EC and99mTc-DMSA are shown in Fig. 11. In

TLC using acetone as solvent, the com-

plexes remained at the point of applica-

tion. Under identical conditions,99mTcO4

- moved towards the solvent

front. However, if hydrolyzed techne-

tium (99mTcO2) is present, it will also

remain at the origin and hence an addi-

tional quality control procedure was

essential to estimate this radiochemical

impurity in the complex. In the paper

chromatography patterns, using 50%

acetonitrile in water solvent for 99mTc-

EC and 0.9% saline solution for 99mTc-

DMSA, both free 99mTcO4- as well as

the complexes migrated towards the

Table 3. Elution performance of the generator

ElutionNo.

99Mo activityGBq (mCi)

99mTc growthperiod (h)

Expected 99mTcactivity GBq (mCi)

99mTc recoveredGBq (mCi)

%99mTcrecovered

1 9.25 (250.1) 24 5.8 (157.2) 5.3 (143) 912 7.2 (194.3) 24 4.5 (122.7) 4.2 (112.9) 923 5.5 (150.1) 24 3.5 (94.7) 3.1 (84.3) 894 4.3 (116.6) 24 2.7 (73.6) 2.4 (66.24) 905 3.3 (90.6) 24 2.1 (57.2) 1.8 (49.7) 876 2.6 (70.4) 24 1.6 (44.5) 1.4 (39.1) 887 1.6 (44.5) 48 0.87 (23.6) 0.71(19.3) 828 0.99 (26.8) 24 0.62 (16.9) 0.52 (14.2) 849 0.77 (20.8) 24 0.49 (13.2) 0.41 (11.2) 85

0 1 2 3 4 5 6 7 8 9 100

2000

4000

6000

8000

10000

12000

14000

99mTcO2

99mTcO4

-

Act

ivit

y (c

pm)

Distance from the point of application (cm)

Fig. 9. Paper chromatographic pattern of99mTc in 85% methanol medium

0 500 1000 1500 2000 2500 3000 3500 4000

2000

4000

6000

8000

10000

1200099mTc: 140 keV

Act

ivit

y (C

PM

)

Channel number

Fig. 10. c-ray spectrum of 99mTc

Original Chromatographia 2010, 72, November (No. 9/10) 881

solvent front, whereas hydrolyzed tech-

netium remained at the origin. By com-

bining the results of both the paper

chromatography patterns, the complex-

ation yield was estimated to be >98% in

both cases. These results justify the

suitability of 99mTc obtained from the

generator for radiopharmaceutical

applications.

Recovery of Mo from theSpent Generator andRegeneration of the Column

In view of the fact that expensive en-

riched 98Mo target needs to be irradiated

for the preparation of a clinical scale99Mo/99mTc generator, this process

would be more economically viable if the98Mo adsorbed on the t-ZrO2 matrix

could be recovered and reused in many

cycles of operation. Moreover, since

t-ZrO2 is not widely available commer-

cially, it is also desirable to regenerate

the used generator column after the ex-

piry of its shelf-life. Therefore, attempts

were made to remove the absorbed 99Mo

from the generator column after expiry

of the shelf-life of the generator. Mo

from the exhausted column was recov-

ered using 3 M NaOH solution (15 mL

NaOH solution + 1 mL 30% H2O2)

and the elution behavior of Mo, as fol-

lowed by counting radioactivity in the

effluent is shown in Fig. 12. It is indi-

cated that the recovery is quite fast as at

the beginning and all the Mo could be

eluted out in the first 3–4 mL of the

eluent. At 3 M NaOH concentration,

t-ZrO2 desorbed >95% of Mo. After

regeneration of the column, 99Mo activ-

ity was reloaded in the column. The

separation efficiency and quality of99mTc obtained from the regenerated

column remained unaltered.

The excellent properties exhibited by

t-ZrO2 indicated that it could be used in

the preparation of 99Mo/99mTc generator

for biomedical applications.

Discussion

The fission 99Mo supply chain has been

severely hit by the cascade of events in

2008 [35]. Although this crisis has now

passed, this has not only highlighted the

fragile nature of fission 99Mo supplies,

but also raised concerns over its depen-

dence. In order to overcome these

unforeseeable problems and to reduce

reliance on fission-produced 99Mo,

alternative pathways in short-term and

long-term requirements need to be

emphasized. Alternative options for long

term production of 99Mo include aque-

ous homogeneous reactors (AHR) con-

cept [36], high-power accelerator and

photo fission of 238U [37]. Although such

approaches hold promise, considerable

research and huge resources are required

to develop, test and deploy the technol-

ogy. The economics of these routes will

remain debatable, and these approaches

are not likely to materialize in the near

future. Concerted efforts are necessary to

explore the possibility of accessing 99mTc

from (n, c)99Mo using high capacity

sorbents that can contribute to the re-

gional needs. In this context, the scope of

using nanomaterial based sorbents such

as tetragonal nano-zirconia (t-ZrO2)

material seemed appealing due to the

high specific surface area and high sur-

face reactivity. This approach can sup-

plement the current production capacity

to a reasonable extent in the future. Our

investigation mostly centers on this

objective.

It is of interest to test t-ZrO2 for the

preparation of 99Mo/99mTc generator for

clinical applications as the material is

non-toxic, chemically inert and ther-

mally stable. The salient features of the

adapted synthesis protocol are (i) nano-

sized oxides (<10 nm) can be prepared

at low temperatures, (ii) the products are

homogeneous and have high surface

area, (iii) the particles are porous and

exhibit excellent flow characteristics. An

important property of nano-zirconia is

its extremely low solubility in dilute

mineral acids and alkalis which is crucial

for practical application. The material is

capable of preserving its physicochemi-

cal and sorption properties in long use.

Batch experiments were pursued to study

the sorption behavior of 99Mo and 99mTc

ions on t-ZrO2. The quick attainment of

equilibrium indicates that the sorption

sites are well exposed on the surface of

this kind of material. The distribution

ratios (Kd) of99Mo and 99mTc at differ-

ent pH were evaluated in order to predict

the experimental conditions necessary

for the optimal loading of 99Mo as well

as efficient elution of 99mTc.

The surface acid–base properties of t-

ZrO2 are inherent and primarily

responsible for the retention of 99Mo.

The surface association of 99Mo ions

with sorbent is governed by the pH of

the external solution, since it determines

the surface charge of solid particles and

degree of ionization. The dependence of

zeta potential (f) on the pH of the

external solution, helps in finding the

(a)

0 1 2 3 4 5 6 7 8 9 100

2000

4000

6000

8000

10000

12000

14000

16000

18000

20000

99mTcO4

-

99mTc-complex99mTcO

2

Act

ivit

y (c

pm)

Distance from the point of application (cm)

99mTc-DMSA

99mTc-EC

0 1 2 3 4 5 6 7 8 9 100

2000

4000

6000

8000

10000

12000

14000

99mTcO2

99mTc-complex99mTcO

4

-

Act

ivit

y (c

pm)

Distance from the point of application (cm)

99mTc-DMSA

99mTc-EC

(b)

Fig. 11. Paper chromatographic pattern of99mTc-DMSA and 99mTc-EC complexes devel-oped in a acetone medium b 50% acetonitrilein water solvent for 99mTc-EC and 0.9%saline solution for 99mTc-DMSA

0 1 2 3 4 5 6 7 8 9 10 11 120

10

20

30

40

50

60

% 99

mT

c el

uted

Volume of NaOH (mL)

Fig. 12. Recovery of Mo from the usedcolumn

882 Chromatographia 2010, 72, November (No. 9/10) Original

sorption mechanism. The pHpzc was

reached at *pH 4.5. In aqueous sys-

tems, t-ZrO2 particles are hydrated, and

:Zr-OH groups cover their surface

completely. These amphoteric hydroxyl

groups can undergo reaction with either

H+ or OH- and develop positive or

negative charges on the surface depend-

ing on the pH [38]. It was observed that

the t-ZrO2 surface has a positive charge

at pH <4.5 and it reversed at pH >4.5.

At pH <4.5, a positive charge density

builds up on the surface of the t-ZrO2

� Zr�OHsurface þHþaq�

� Zr�OHþ2surface

At weakly acid solution, molybdate

polymerizes as follows [39]:

7MoO2�4 þ 8Hþ�Mo7O

6�24 þ 4H2O

There is a strong attractive interac-

tion between the positively charged

t-ZrO2 surface and the negatively

charged species from the liquid solution.

The mechanism of 99Mo uptake there-

fore may be considered to take place in

two steps. The first one may be the

exchange of polymolybdate anions for

hydroxyl ions on the surfaces of the

particles. Subsequently, it may form a

stable complex of the type [ZrMo6O24]8-,

similar to that reported with alumina

[40]. The decay of 99Mo to 99mTc is not

accompanied by any serious disruption

of chemical bonds. As these polymolyb-

date ions start transforming into per-

technetate ion (99mTcO4-), which has

only -1 charge, the binding would get

weaker and the 99mTcO4- gets easily

eluted with normal 0.9% saline. How-

ever, in order to predict the exact mech-

anism of 99Mo uptake, further studies are

warranted which is beyond the scope of

this work.

The sorption capacity, indicative of

the ability of t-ZrO2 to retain 99Mo,

depends on the number of available

sorption reactive sites per unit mass

within the solid matrix. In the present

investigation, the dynamic sorption

capacity was seen as a criterion for

retention capacity of the sorbent in the

point-of-use system. It may be noted

that the practical sorption capacity

(155 ± 12 mg Mo g-1) of t-ZrO2 is

significantly higher than that of alumina

(2–20 mg Mo g-1). However, by allow-

ing the 99Mo solution to load under

static conditions in a batch reaction,

a sorption capacity of *250 ±

10 mg Mo g-1 could be achieved. The

protocol for preparing chromatographic

column using sorbent matrix preloaded

with 99Mo is not recommended owing to

difficulties in handling of radioactive

material. This will not only cause asso-

ciated radioactive contamination prob-

lem but also increase the radiation

exposure to the personnel involved.

Furthermore, even if the column is pre-

pared, the operating performance there-

of is likely to deteriorate. Therefore, post

loading of 99Mo activity protocol, was

followed despite the lower capacity

(155 ± 12 mg Mo g-1) that could be

achieved.

The nano-oxide was endowed with

considerable specific surface area and

the porous framework facilitated acces-

sibility to more reactive sites with

pronounced adsorption affinities. Con-

sequently, the 99Mo retention capacity is

significantly high which represents an

important advantage and would facili-

tate use of (n, c) 99Mo on the column

while concurrently allowing elution of99mTc in high radioactive concentration.

The chromatographic column contain-

ing t-ZrO2 was validated for 10 days

using 9.25 GBq (250 mCi) of 99Mo to

evaluate the behavior of the system in

the presence of intense radiation envi-

ronment with the radiolytic products

generated as a result of radioactive99Mo. The elution profile obtained was

sharp and high radioactive concentra-

tion of 99mTcO4- could be obtained.

Reproducible recovery of 99mTc was

observed with a yield >85%. The pur-

ity, elution yield of the 99mTcO4- and

99Mo breakthrough remained nearly

unchanged with long term operation.

Radiolabeling studies were carried out as

they provide information about the

quality of the separated radiotracers to

form labeled compound in nano-molar

concentrations. The performance of the

generator at higher activity level

(185–370 GBq) would be a real test of

the radiation stability of the sorbent. Use

of enriched 98Mo target and high flux

reactors would enable obtaining high

specific activities of 99Mo. Owing to the

non-accessibility of high flux reactors

and unavailability of enriched 98Mo target

at our institution, such experiments

could not be initiated and shall be taken

up in due course. However, the present

findings amply suggest that this material

holds promise for use as a sorbent

for the preparation of a clinical grade99Mo/99mTc generator.

When the activity of the 99Mo

decreases below a certain value, the99Mo/99mTc generator is no longer useful

for medical applications. However, it

cannot be discarded without removing

the sorbed 99Mo from the column.

Hence, procedures for removing 99Mo

from the column prior to disposal were

pursed. Owing to the reversible nature of

the interaction between t-ZrO2 and99Mo

ions, it could be released from the solid

matrix under alkaline condition. This

step is also important, if Mo recovery is

needed in case the procedure is adopted

with enriched target. Moreover, t-ZrO2

can also be used as a recyclable sorbent.

It is envisaged that the developed

method would serve in good stead for

ensuring the routine daily elution of99mTc using (n, c) 99Mo. Further

research can be focused on the develop-

ment of an automated system.

Conclusions

The potential utility of t-ZrO2 as a new

generation effective and efficient sorbent

for the preparation of 99Mo/99mTc gen-

erator using (n, c) 99Mo was demon-

strated and appears to offer good

prospects. 99mTc could be obtained in

>85% yields with high radionuclidic and

radiochemical purity. The compatibility

of the product in the preparation of99mTc labeled formulations was evalu-

ated and found to be satisfactory. With

the advantages of simple synthesis pro-

cedure, excellent 99Mo binding capacity

and good chromatographic performance,

t-ZrO2 is a promising candidate for the

preparation of clinical scale 99Mo/99mTc

generator of moderate strength using

medium to low specific activity (n, c)99Mo. The present concept would be of

Original Chromatographia 2010, 72, November (No. 9/10) 883

considerable value for accessing 99mTc,

particularly in countries with no facilities

for fission 99Mo production.

Acknowledgements

The authors are grateful to Prof. V. Ve-

nugopal, Director, Radiochemistry and

Isotope Group, BARC for his valuable

suggestions during the course of the

work.

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