Simpler Less Expensive Method for Analysis of inorganic As (iAs) in rice
Rufus L. Chaney1, Carrie E. Green1, Steven J. Lehotay2 and Michael Bukowski3
USDA-Agricultural Research Service
Crop Systems and Global Change Laboratory, Beltsville, MD.
2
Eastern Regional Research Center, Wyndmoor, PA.
3
Grand Forks Human Nutrition Laboratory, Grand Forks, ND.
1
Project Goal: Develop a reliable, simple, more rapid and less expensive method for the
analysis of inorganic As (iAs) in rice grain. We undertook three major sub-projects to meet
these goals:
1.
Develop and validate a simple reliable lower cost method to measure iAs in rice than the
US-FDA method using HPLC-ICP-MS for full As speciation.
2.
Subject the developed method to an Inter-Laboratory Evaluation to determine if it was
as reliable as observed in our lab, and could be conducted easily by other scientists.
3:
Attempt to develop a more rapid extraction method to reduce the time needed to extract
iAs from powdered rice and analyze the iAs in those extracts.
SUMMARY:
New limits on iAs in rice products require that samples be analyzed for iAs to assure
compliance. Initially reported methods (high pressure liquid chromatography-Inductively coupled
Plasma-Mass Spectrometry, HPLC-ICP-MS) used measurement of all species of As present in
rice and other foods, which requires very expensive staff and equipment, and a high cost per
sample for rice iAs analysis. Industry needs a reliable less expensive method to measure the
needed iAs, not full As speciation, in order to comply with market limits. We developed and
then conducted an Inter-Laboratory Evaluation of a simple Hydride Generation (HG) method to
measure iAs directly rather than as part of As speciation. Arsenate and arsenite (iAs) in the
solution from the US-FDA method to extract iAs from powdered rice [90 min. at 95°C in 0.28 M
HNO3] was pre-reduced to arsenite in a stronger HCl solution before HG analysis so that the
HG method was capable of minimizing measurement of dimethylarsinic acid (DMA) present in
rice extracts. The HG method can use much less expensive analytical instruments, and be
conducted by normal laboratory staff much more rapidly than the US-FDA full As speciation
method. To conduct the Inter-Laboratory Evaluation , samples of brown and milled rice with low,
1
medium and high levels of iAs were identified and prepared. Following the comparison protocol
blind duplicate samples of these samples were sent to the cooperating laboratories and most
labs conducted triplicate extraction and analysis of the test samples and the NIST standard rice.
Four labs conducted the HPLC-ICP-MS analysis for comparison with the HG method (two labs
did both methods). Most laboratories obtained correct iAs results for all unknown rice samples
using the HG method.
Attempts to use simple shaking of rice powder with several extraction solutions were
partially successful in that neutral pH H2O2 solutions could release the rice iAs, but filtering the
extracts took much longer (~3 hr) and defeated any benefit compared to HotBlock extractions.
Additional testing of the time required for the HotBlock extraction of iAs showed that full
extraction with small variance could be achieved in 15-30 min. rather than the 90 min. in the
US-FDA method. Although methods for analysis of truckload samples before delivery appear
unnecessary because nearly all US rice complies with CODEX iAs limits, it may be possible to
dehull, mill, grind, extract and analyze iAs in such samples using the shorter HotBlock
extraction and the HG iAs analysis method with suitable inexpensive analytical equipment if
trucks continue to wait long periods before delivering their loads.
BACKGROUND:
This project focused on inorganic As (iAs) rather than total As (TAs) because it is only
the iAs that has been shown to contribute to the adverse health effects of dietary As (US-FDA,
2016a). Because iAs exposure is an important problem around the globe, research on iAs
presence and bioavailability continue outside of the interest about iAs in rice and rice products.
Rice iAs Measurement Methods:
Many methods have been used to extract and measure As concentrations and species
in rice. A review by Welna et al. (2015) summarizes many attempts to evaluate total As and As
species in rice and other foods. With the increasing recognition that iAs was the important As
species that would be regulated, researchers looked for methods to reliably extract and then
analyze iAs. Different scientists tested iAs extraction using enzymatic methods, method using
trifluoroacetic acid, dilute HNO3, methanol, and other reagents. Over time the use of the present
US-FDA method with 0.28 M HNO3 (Kubachka et al., 2012) has been widely adopted. This
method is similar to the extraction approach used in an European Union collaborative study of
methods to measure iAs in rice, a method needed before the iAs level could be regulated (de la
2
Calle et al., 2011).
Thus, the official US-FDA method to analyze iAs in rice uses a HotBlock [a heating
block with computer control and uniform temperature which can process many samples (47) at
one time] extraction of As species from rice powder using 0.28 M HNO3 (1 g rice with 10 mL of
HNO3), followed by centrifugation, membrane filtration and pH adjustment to support use of
high performance liquid chromatographic separation (HPLC) of the extracted As species.
Inductively Coupled Plasma-Mass Spectrometry (ICP-MS) [a high end mass spectrometry
system is normally used for this analysis, a triple-quadrupole ICP-MS (ICP-QQQ) using oxygen
as reaction gas in a collision cell and detection of AsO at m/z 91] serves as the detector for
separated As species (Kubachka et al., 2012). Because analysis of arsenite can be confounded
by DMA-As, others have included 1% hydrogen peroxide (H2O2) to oxidize all iAs to arsenate
which is more readily separated from DMA in the HPLC-ICP-MS method (e.g., Pétursdóttir et
al., 2014; Raber et al., 2012). ICP-MS or ICP-MS-MS instruments to do these analyses cost
$150,000 to $300,000, and maintenance and operation requires a highly trained analyst.
However, this complicated system is not needed if only iAs is the desired measurement. The
HotBlock digestion method used by US-FDA is widely accepted for extraction of As species
from powdered rice, and with a $6,000 HotBlock device, one can extract 47 samples in a half
day of work (one position in the Block is used for the temperature control system). To measure
total As in rice, use of a microwave digestion device which can operate up to 270°C is often
needed which costs $30,000-70,000 and processes 15-40 samples at a time. Cleaning
equipment for the microwave digestion apparatus between samples is also time consuming.
It is now evident that the analytical result needed to comply with any limits for iAs in rice
(US-FDA, 2016) is simply the amount of iAs in samples of finely ground rice, not the full As
speciation. Many methods (Welna et al., 2015) have been reported in the literature, but
application for analysis of the typical levels of iAs in rice limit their use. Thus, we conducted
research to develop a simple Hydride Generation (HG) method to measure the iAs in the
extract from the HotBlock digestion using 0.28 M HNO3. Our earlier testing of sample
preparation methods for TAs analysis by HG had revealed that the microwave digestion used to
release all of the As (TAs) from powdered rice had to achieve about 270°C for at least one hour
to hydrolyze the dimethylarsinic acid (DMA) which is the main organic form of As in rice.
Digestion at 225° (maximum we could attain with an older microwave digestion instrument) did
not hydrolyze DMA, so HG analysis of those lower temperature digestions gave results
essentially equal to the iAs in NIST rice Standard Reference Material (SRM) (not the total As).
Separately from the present work we had tested dry ashing to measure the As in powdered rice
3
but found that ashing at 480°C removed the DMA component in total As, so we had to use a Mg
ashing aid of an old AOAC method for analysis of total As in feedstuffs (AOAC, 1965). Thus it
appeared that short of high temperature microwave digestion, one could release the iAs and
analyze the iAs using a simple hydride generation (HG) approach rather than the usual FDA
HPLC-ICP-MS method. [An alternative As speciation method has been reported by Jackson
(2015) which uses different resins to separate the As species in HPLC-ICP-MS analysis of rice
As so that the run time is 2-5 min. rather than the 20 min. of the original US-FDA method
(Kubachka et al., 2012).] But the high end instrument and staff needed for the analysis does not
reduce the high cost of that approach to obtain the iAs concentration.
HG can be used with many different measurement technologies, not just ICP-MS, and
the staff needed to conduct those analyses with other instruments do not require such a high
staff training level to achieve reliable results. The arsine gas generated by the HG treatment of
a HotBlock digestion solution can be measured by ICP-atomic emission spectrometry (ICPAES), by ICP-MS, by Atomic Absorption Spectrometry (AAS) or Atomic Fluorescence
Spectrometry (AFS), allowing labs to use lower cost equipment and staff to conduct rice iAs
analyses.
With support from The Rice Foundation, we undertook to further develop the simple
HotBlock-HG-ICP-AES method to measure iAs in rice [see Standard Operating Procedures for
Extraction of iAs from powdered rice (Appendix 1), and HG-ICP-AES analysis of iAs in rice
digests (Appendix 2)]. Based on research by Feldmann’s group (Musil et al., 2014; Pétursdóttir
et al., 2014), it appeared that using higher concentration of HCl in the sample subjected to HG
could strongly reduce the potential for DMA in an extract of rice to be converted to arsine and
be measured by the atomic spectrometric methods. They had not pre-reduced sample arsenate
(present because H2O2 was included in their extraction fluid) before using the HG method, while
we had followed methods which pre-reduced arsenate before HG was conducted (the US-FDA
extraction does not include H2O2). We did extensive checking of the effect of HCl concentration
on measurement of iAs vs. DMA-As in extracted rice solutions from the HotBlock. Spiking the
rice extracts with varied levels of DMA and iAs, and testing varied HCl concentrations, with both
milled and brown rice with varied levels of iAs, we confirmed the finding of Pétursdóttir et al.
(2014) but using “pre-reduced” solutions in the current method (Figure 1). The “carry-over” of
As from DMA (or error from DMA) was reduced to about 4% of the DMA-As present in a
sample, so the possible error from using HG to make the measurement was smaller than the
usual variability of analysis of rice samples using HPLC-ICP-MS. If the iAs approaches the
4
Figure 1. Effect of HCl concentration in the pre-reduction solution on measurement of DMA-As
in addition to the iAs present. We selected 4 M HCl for the method to protect the equipment
from acid vapors. Different color lines show results from different rice samples and DMA spike
levels.
CODEX limit of 200 ìg iAs kg-1 fresh weight of rice, one could analyze the total As, subtract the
measured iAs, and then subtract 4% of the remaining As which came from DMA to correct the
iAs result.
The method using HotBlock digestion, filtration, and HG analysis using ICP-AES was
studied further to simplify it as much as practicable and minimize costs without increasing
variance in analysis. For HG analysis, it is not critical use centrifugation and millipore filtration of
each sample (needed to protect HPLC and ICP-MS equipment); simple filtration with filter paper
and funnel yields sample solution ready for HG analysis.
At this point, the method was ready for an Inter-Laboratory Evaluation. Dr. Steven
Lehotay, USDA-ARS-Eastern Regional Research Center, an analytical chemist who has
previously conducted Inter-Laboratory Evaluations of various analytical methods, agreed to
officially steer the Inter-Laboratory Evaluation study. He designed the set of test samples to be
supplied to cooperating labs based on the standard AOAC International method evaluation
template for collaborative studies across labs. We purchased and otherwise obtained rice bulk
5
samples and analyzed them for TAs and iAs to obtain a range of TAs and iAs in both milled and
brown rice. Dr. Lehotay selected specific large samples containing low, medium, and high levels
of iAs for use in the study (Tables 1A, 1B). We then contacted more than 20 laboratories
internationally which have conducted As analysis for environment and food samples, especially
rice, and invited their participation in the Evaluation. We sent known liquid standards, NIST
Standard Reference Material (SRM 1568B) rice flour sample, and 12 unknown powdered rice
samples (randomized blind duplicate samples of 3 brown and 3 milled rice samples with low,
medium and high iAs levels) to each of the labs (Table 2) which volunteered to participate.
Several other labs asked to participate after hearing about the Evaluation being conducted; 14
returned the results of their analyses, two of which used only the FDA method, one used both
the FDA and HG methods, and one analyzed different portions of the test samples twice by the
HG method using different detection systems. A preliminary statistical evaluation of the InterLaboratory Method Evaluation is reported below; preliminary results without removal of possible
outliers show acceptable results based on the Horwitz Ratio (Horwitz and Albert, 2006).
Table 1A. Samples provided to Laboratories
1
2
3
4
5
6
7
8
9
10
11
12
sample 600 - double golden
row 40 brown
CLXL 745 flood white
row 40 brown
row 40 white
sample 400 - Tsuru Mai
CLXL 745 flood brown
row 40 white
CLXL 745 flood white
sample 600 - double golden
sample 400 - Tsuru Mai
CLXL 745 flood brown
Table 1B. Structure of Blind Duplication of Samples.
1+10
2+4
3+9
5+8
6+11
7+12
sample 600 - double golden
row 40 brown
CLXL 745 flood white
row 40 white
sample 400 - Tsuru Mai
CLXL 745 flood brown
Interestingly, several of the labs using ICP-MS to measure the arsine As from the HG
method had difficulty in measuring As using HG of arsine. We asked Dr. J. Feldmann’s team
6
Table 2. List of laboratories which volunteered to participate in the Inter-Laboratory evaluation
of the simple HG method to measure iAs in powdered rice.
Dr. Tomohito Arao
HG-ICP-AES
Principle Research Coordinator
National Institute for Agro-Environmental Sciences
Tskuba, Ibaraki 305-8604
Japan
Dr. Michael Bukowski
USDA-ARS-GFHNRL
PA-3062
2420 2nd Avenue North
Grand Forks, ND 58203
HG-ICP-MS and HG-AAS
Dr. Rufus L. Chaney
USDA-ARS-NEA-BARC-CSGCL
Bldg. 007, Room 013
Beltsville, MD 20705
HG-ICP-AES
Dr. Sean Conklin
Chemical Contaminants Branch
US-FDA/CFSAN/ORS/DBC
Food and Drug Administration
5100 Paint Branch Parkway, HFS 706
College Park, MD 20740
HPLC-ICP-MS only
Dr. Jörg Feldmann
HG-ICP-MS and HPLC-ICP-MS
Chair in Environmental Analytical Chemistry
TESLA- Trace Element Speciation Laboratory
University of Aberdeen
Meston Building Rm G26
Aberdeen AB24 3UE
Scotland UK
Dr. Kent Lanclos
HPLC-ICP-MS only
Technology and Science Division
USDA-Grain Inspection, Packers, and Stockyards Administration
National Grain Center
10383 N. Ambassador Drive
Kansas City, MO 64153
7
Dr. Won-Il Kim and Dr. Anitha Deepak
HG-ICP-MS
Head, Department of Agro-Food Safety
Rural Development Administration (RDA)
National Academy of Agricultural Science (NAAS)
Korea
Dr. Andrew A. Meharg
Institute for Global Food Security
Queen’s University Belfast
David Keir Building
Malone Road
Northern Ireland
UK BT9 5BN
HPLC-ICP-MS only
Dr. Philip Moore
USDA-ARS; Plant Sciences 115
University of Arkansas
Fayetteville, AR 72701
HG-ICP-AES
Dr. Trenton L. Roberts
HG-ICP-AES
Dept. Crop, Soil, and Environmental Sciences
1366 W. Altheimer Drive
Fayetteville, AR 72704
Dr. Angelia L. Seyfferth
HG-ICP-MS
Dept. of Plant and Soil Sciences
531 South College Avenue; Townsend Hall 152
Dept. of Plant and Soil Sciences; University of Delaware
Newark, DE 19716
Cheryl D. Stephenson
Laboratory Director
Eurofins Central Analytical Laboratories
2219 Lakeshore Drive, Suite 500
New Orleans, LA 70122
HG-ICP-MS
Dr. M.H. Wong [[email protected]] HG-ICP-AES
Department of Science and Environmental Studies,
Hong Kong Institute of Education, Tai Po,
Hong Kong,
PR China
Dr. F.-J. Zhao
HG-ICP-AES
College of Resources and Environmental Sciences
Nanjing Agricultural University
Nanjing 210095, China
Dr. Y.-G. Zhu
HG-ICP-MS
Research Center for Eco-Environmental Sciences
Chinese Academy of Sciences
Beijing, 100085, China
8
how they were able to successfully link the HG gas stream into ICP-MS and we shared their
information with several participants who had difficulty with the connection. All but one of these
other labs were able to obtain accurate results. That problem delayed several labs from
completing the test. One lab had great difficulty in hooking HG into their ICP-AES and did not
obtain valid results, while other labs which used Perkin-Elmer or Agilent ICP-MS instruments
were able to do well with these samples using HG. As part of the development of the simple
method to measure iAs in powdered rice, we prepared an estimate of the cost of materials and
staff to conduct the analyses (Table 3). We did not include instrument or lab space costs
because those vary among locations, but the supplies and labor costs are fundamental to any
labs cost for analysis of rice samples. The present method uses less expensive equipment and
staff to attain the needed iAs result, providing great savings to the rice industry.
Table 3. Estimated Cost of Rice iAs Analysis.
Task
Cost -Labor & Supplies
Milling brown rice sample
?
Grinding milled rice sample
200 samples/8 hr
$1.41/sample
Hot Block extraction of iAs
113 unique rice samples/8 hr
$2.75/sample
HG-ICP-AES analysis of iAs
59 unique rice samples/8 hr
$2.85/sample
Management; Report Preparation
?
59 unique rice samples/8 hr day/HG-ICP-AES method
Equipment, ICP-AES, laboratory space and management costs not included in estimates.
BS-level trained staff can conduct all of the operations involved. MS/Ph.D. to supervise,
maintain, ICP-AES, evaluate QA, and prepare reports.
Tables 4A and 4B list the iAs results using the HG method for 10 labs in 5 countries.
Two labs were very late to report their results and demonstrated problems in the analyses,
including apparent mismatching of samples. Thus their results were not included in the
assessment with clear justification. Table 4B shows the Horwitz Ratio evaluation of these
analyses.
In comparison, the results for the same samples are presented from the FDA HPLCICP-MS method performed by four labs are reported in Tables 5A and 5B. The determined
9
concentrations were within 13% of each other in all cases, and all Horwitz Ratios were also
<0.67. Inter-laboratory trials with >8 participating labs and Horwitz Ratio <2 are generally
acceptable as an AOAC International Official Method after statistical expert panel review. A
Horwitz Ratio of 1 is the average among previous Official methods, and a value <1.0 indicates
better than average, which is the case in this study even before outliers are removed. The
formality of attaining AOAC Official Method status will not be pursued due to prohibitive cost
and time for AOAC International approval, but the scientific statistical assessment shows that
the method meets the common method acceptance criteria. A manuscript will be prepared for a
peer-reviewed journal to report the Inter-Laboratory trial.
Four labs provided the FDA method results (HPLC-ICP-MS) for iAs analysis of the rice
test samples. Table 5A shows the results from each of the labs for each sample. Table 5B
shows the means of the blind duplicate samples from each lab, and the Horwitz Ratio
calculation for the HPLC-ICP-MS method.
Figure 2 shows a direct comparison of the mean results for each sample for both
methods. The Figure shows that the HG-ICP-AES method results were not significantly
different from the HPLC-ICP-MS method results, illustrating that the methods yield equivalent
results. Table 6 reports the statistical comparison of the HG-ICP-AES and HPLC-ICP-MS
methods.
10
Table 4A. Analyses of samples from cooperating laboratories. Samples arranged in pairs of blind duplicates for easier comparison of within lab
variation.
Sample N
Mean
S.D.
RSD
Horwitz
Ratio
1
2
3
Cooperating Laboratory Number
4
5
6
7
8
9
10
1
10
10
10
77
76
5
9
7%
12%
0.29
0.52
72
90
76
76
84
90
82
70
70
64
73
69
87
76
74
62
79
81
76
81
2
4
10
10
133
135
16
15
12%
11%
0.55
0.53
107
109
139
136
145
152
140
141
121
120
132
128
148
150
106
113
146
148
150
149
3
9
10
10
181
182
24
20
13%
11%
0.65
0.55
156
195
184
187
222
202
215
212
151
151
174
166
197
190
149
146
180
190
185
179
5
8
10
10
93
93
11
10
12%
11%
0.52
0.48
76
85
93
93
104
97
100
99
80
82
92
85
107
100
77
74
100
105
101
106
6
11
10
10
125
125
16
15
13%
12%
0.59
0.54
119
123
128
127
138
142
135
137
105
109
113
116
143
136
93
92
141
136
133
131
7
12
10
10
254
255
29
38
11%
15%
0.58
0.76
235
233
265
265
291
310
291
298
220
225
225
247
260
271
207
168
279
268
263
265
NIST
10
108
12
12%
0.52
109
112
111
131
89
98
111
96
97
124
Table 4B. Means for the blind duplicate samples analyzed by 10 cooperating labs which used the Hydride Generation (HG) method compared with
mean for all labs for HG method.
Samples
N
Mean
SD
1+10
2+4
3+9
5+8
6+11
7+12
10
10
10
10
10
10
------------------------------------------------------------------ng g -1 -------------------------------------------------77
6
81
76
87
76
67
71
81
68
80
79
134
15
108
137
149
140
120
130
149
110
147
149
182
21
175
186
212
214
151
170
194
148
185
182
93
10
80
93
101
99
81
88
103
76
103
104
125
15
121
127
140
136
107
115
140
92
139
132
254
33
234
265
301
295
223
236
265
188
274
264
0.33
0.53
0.56
0.48
0.56
0.66
NIST
10
108
0.52
12
1
109
2
112
3
111
11
4
131
5
89
6
98
7
111
8
96
9
97
10
124
Horwitz Ratio
Table 5A. Results of analysis of blind duplicate samples of 4 cooperating labs which used the
HPLC-ICP-MS (FDA) method.
Sample
N
Mean
SD
RSD
Horwitz
Ratio
ng iAs g-1
1
Cooperating Lab
2
3
4
-------- ng iAs g-1 --------
1
10
4
4
85
82
9
8
11%
10%
0.48
0.42
96
86
81
76
92
93
72
73
2
4
4
4
154
148
14
14
9%
9%
0.43
0.45
169
155
152
153
162
161
132
124
3
9
4
4
203
196
27
23
13%
12%
0.65
0.59
236
211
181
193
222
220
172
159
5
8
4
4
106
107
10
10
9%
10%
0.42
0.43
119
118
97
102
113
116
96
93
6
11
4
4
140
134
13
15
9%
11%
0.43
0.51
148
147
137
131
154
148
120
111
7
12
4
4
282
282
28
25
10%
9%
0.51
0.46
296 278
301 269
314
309
239
248
22%
NIST
4
108
15
0.62109
99
133
14%
Table 5B. Within lab means for duplicate samples analyzed by HPLC-ICP-MS method
Samples
1
2
3
4
Horwitz Ratio
1+10
2+4
3+9
5+8
6+1
7+12
75
162
224
119
147
299
69
153
187
100
134
274
81
161
221
114
151
311
64
128
165
94
116
243
0.38
0.43
0.60
0.42
0.47
0.48
12
93
Figure 2. Comparison of inorganic As (iAs) concentration (ng g-1) in 6 rice test samples
measured by the HG-ICP-AES method compared with results from the the HPLC-ICP-MS
method (FDA method).
Table 6. Statistical comparison of the HG and FDA methods to measure iAs in rice.
FDA Method
HG Method
Duplicate
Samples N
Mean
SD
1+10
2+4
3+9
5+8
6+11
7+12
6
6
6
6
6
6
68.6
152.7
187.2
99.8
134.3
273.7
4.6
2.2
7.2
5.1
6.7
8.2
6.8%
1.4%
3.8%
5.1%
5.0%
3.0%
1+10
2+4
3+9
5+8
6+11
7+12
6
6
6
6
6
6
79.9
147.0
184.8
102.7
138.5
273.5
2.7
4.0
5.1
3.1
3.2
7.6
3.4%
2.7%
2.8%
3.0%
2.3%
2.8%
11.3
-5.7
-2.3
2.8
4.2
-0.2
NIST
3
99.2
1.9
1.9%
NIST
3
97.4
3.8
4.0%
-1.7
RSD
Duplicate
Samples N
13
Avg
SD
RSD Diff (ng/g)
Testing possible approaches for rapid extraction of iAs and Cd from powdered rice:
A second goal of this project was to find a simpler method to rapidly extract iAs from
ground rice and measure the iAs released. The possible need for Cd analysis of rice samples
became evident as it was learned that the most important agronomic practice to reduce iAs in
rice would be to use Alternative Wetting and Drying (AWD) so that rice soils were more aerobic
such that less arsenite was produced during growth, thereby lowering the amount of As
absorbed and translocated into rice grain. But while making the soil more aerobic reduced grain
iAs, it automatically caused increased grain Cd (Arao et al., 2012; Chaney, 2015; 2016; Linquist
et al., 2014; LaHue et al., 2016; Moreno-Jiménez et al, 2014).
Rapid extraction of iAs in rice grain needed to take into account that much of rice iAs
is arsenite chemically linked to sulfhydryl groups in the rice proteins (Lombi et al., 2009; Carey
et al., 2012; Meharg et al., 2008). This chemical speciation of As in rice materials is important in
discovery of methods to release and measure the iAs important to regulation of rice and rice
products.
Initially we considered that such a rapid extraction-analysis method could be used to
evaluate rice in truckloads arriving at rice mills so that the loads of rice could be segregated
based on their iAs concentration. However, a visit at the facilities of a major rice mill and their
labs where grower-delivered rice samples were evaluated, at the Grain Inspection and
Stockyards Administration (GIPSA) lab facility in Stuttgart, AR, and at the ARS labs and
scientists at Stuttgart, AR, made it clear that this goal may not be needed or practical for the
rice industry. Presently rice mills sample each truckload delivering their crop at the mill, and
combine the truckload samples from particular fields or cultivars from one grower into a single
“lot” of rice for evaluation of the moisture, milling properties and ultimately the cash value of the
delivered rice. Over time the composited samples from one grower/field were processed to
determine the moisture content, milling efficiency and breakage during milling to set the $ value
of the loads from that grower. The threshing/cleaning/husking, milling, and analysis occurred
days after the load was delivered. The drying, threshing, milling and other steps needed to
evaluate the regulatory compliance of a rice load would be very difficult to conduct in a short
period while the truck was in line to deliver a load of rice at a mill. [If an unhulled rice sample
were heat dried to dryness, kernel breakage may be strongly increased during test milling.] It
would be more possible to analyze brown (hulled) rice from a load than milled rice because of
the time needed to achieve valid milling of bulk rice samples from growers. But the ratio of iAs
to TAs in brown rice differs from that of the milled rice which would be generated from that lot of
brown rice. Analytical results for brown rice cannot be used to regulate or make marketing
14
decisions about milled rice (the only form with regulatory limits) (US-FDA, 2016). The reason
that milled rice must be the subject of analysis is that the ratio of DMA-As to iAs varies among
samples due to variation in irrigation practice, soil As and water percolation variation, and rice
genetics which affect As uptake and speciation. Rice bran is much higher and more variable in
TAs and iAs than is the milled rice.
Development of a simpler method to extract the iAs from powdered rice offers value
to the industry if a reliable, simple and less expensive method could be demonstated. We
looked at this extraction in relation to long experience with agronomic soil analysis procedures.
Common soil analysis methods use a simple shaking of weighed soil with known volume of
reagent for a set period, filtration, and analysis. Whether it is P, K, Ca, Zn, Fe or other nutrients,
or pH, a simple extraction followed by a simple analysis gives the needed analyses at low cost
and lower salaried staff.
We looked at reviews of methods to extract and analyze As in rice (e.g., Welna et al.,
2015) which considered many different extraction methods, different reagents, heating or not,
etc., to release the species of As in rice for subsequent full As speciation analysis usually by
HPLC-ICP-MS. This detailed investigation was needed in earlier research in study of rice As
issues, but now that it is clear that only iAs measurements are needed for rice markets, simpler
methods to measure only iAs in rice are clearly what is needed. The separate evidence showed
that much of the As in rice is arsenite chemically bound to sulfhydryls of rice protein (Lombi et
al., 2009). Shen et al. (2013) clarified the normal binding of iAs to proteins in many natural
systems which concluded that when arsenite was bound to proteins it was predominantly
bonded with sulfhydryl groups in the protein.
Based on this knowledge, we hypothesized that shaking powdered rice in solutions of
different sulfhydryl reagents would release the iAs and allow rapid and easy extraction for HGICP-AES analysis of iAs. To test this hypothesis, we extracted rice standard samples with
solutions containing 5 and 10 mM concentrations of cysteine, glutathione and dithiothreitol, at
several pH levels. Analysis of the extracts showed that shaking with sulfhydryl reagents did not
release the iAs for analysis. We checked to assure that the presence of the reagents in the test
solutions did not confound the HG analysis and verified that if these solutions had extracted the
iAs, it would have been measured by the HG-ICP-AES method. We concluded that sulfhydryl
reagents could not release iAs from powdered rice.
Although Shen et al. (2013) discussed use of sulfhydryl reagents (e.g., dithiothreitol)
to release As from proteins, they also reported that previous research had showed variable
release with these reagents. An alternative approach is extraction of the iAs from the protein
15
using H2O2 to oxidize the arsenite to arsenate which is no longer strongly bound to protein-SH
groups and was released for extraction. They cited work by Naranmandura et al. (2006) which
showed ready and rapid release of sulfhydryl-bound arsenite using H2O2 treatment. And work
by Chen et al. (2011, 2013) used reaction with H2O2 overnight to release sulfhydryl-bound As
species into solution so they could be analyzed by HPLC-ICP-MS.
Thus we hypothesized that we should be able to release iAs from powdered rice
using this H2O2 method, and then analyze the iAs by HG-ICP-AES. The optimum pH for this
reaction was not established by previous literature. And because some H2O2 may remain in the
extract, the pre-reduction of arsenate to arsenite before HG analysis needed to be verified.
Testing the effectiveness of H2O2 at acidic and neutral pH showed that little was extracted at
the acidic pH, but near quantitative release was achieved at pH 7. Additional testing was
conducted at pH 6, 7, 8 and 9, and with both milled and brown rice powder to better understand
the effectiveness of this procedure to rapidly release iAs from powdered rice. All of these pH
levels were effective in releasing iAs using 1% H2O2. Using higher levels of H2O2 caused a
major artifact by preventing the rapid reduction of arsenate to arsenite in the “pre-reduction”
step in HG analysis, so the lower H2O2 level which was effective was studied in continuing
evaluation of releasing iAs from powdered rice using a simple shaker method. For those not
familiar with usual shaking extraction methods used in agronomy, we weigh some amount of
soil or powdered rice into a 125 mL urinalysis cup with screw cap lid (very inexpensive so it can
be disposed after one use rather than having to be acid washed between uses requiring
significant time and staff costs). The desired volume of extractant is then added and the cup
placed on a rotating shaker for 30-120 minutes, then filtered using simple filter paper methods
rather than membrane filtration. Inexpensive 1 ounce (28 mL) polyethylene vials are used to
collect the filtrate. Filtration would continue until the bottle was filled or the filer paper was
drained.
Because the suspension of powdered rice in the several extractants tested to date
filtered very slowly, and incompletely, we found using at least 10-20 mL per 1 g of powdered
rice was necessary. If more sample fluid is required to conduct analyses, one could use even
higher amounts.
Although this approach seemed promising initially, it became evident that filtration of
the rice slurry after shaking is very slow in the iAs release testing, taking 1-3 hr for complete
filtration (see Table 7). Long filtration would allow the variation in filtration time among samples
to affect iAs concentration due to variable evaporation of water among samples. More
importantly, an extraction which takes 2-4 hours (weigh; shake; filter) does not achieve the
16
rapid analysis sought for iAs in milled rice samples. Thus we abandoned the shaking approach
because it offered no advantage compared to the HotBlock extraction method. So we examined
that method to see if it could be done more rapidly yet remain accurate.
More rapid extraction using the HotBlock:
When it was evident that simple shaking with H2O2 would not be useful because of
the slow filtration, we re-considered the time requirement for the HotBlock extraction method.
We did not find reports of the relationship of heating time period on extraction of iAs using
HotBlock methods, so we tested the effect of heating period on filtration time and iAs extraction
efficiency. After HotBlock processing, rice samples filter relatively rapidly, and large numbers of
samples can be handled using the HotBlock digestion equipment. Some testing early after
purchase of the HotBlock indicated that rice iAs was released a lot faster than the 90 min. listed
by US-FDA and other researchers who were using HPLC-IC-MS for total As speciation.
Essentially complete extraction of iAs was achieved within 30 min. Thus we evaluated the effect
of time of extraction in the HotBlock for several standard rice samples used in this lab.
The results of these tests are listed in Table 7. Very short HotBlock treatment time
caused slow and inefficient filtration, but 15-30 min. treatment yielded rapid filtration and correct
iAs results. Based on these tests with 4 rice samples, brown and milled, flood and AWD
produced, and the NIST rice 1568B standard, we conclude that all were effectively extracted by
30 min., shortening the needed extraction period and increasing the number of samples that
could be processed in one day by one technician and one HotBlock.
17
Table 7. Effect of time at 90°C in the HotBlock on time for filtration and effectiveness of release of iAs for
several lots of rice (mean ± Standard Deviation for 5 replicate samples).
Rice Sample
Heating Time
Filtration Time
iAs
Std. Dev.
Flood Brown
5
15
30
60
A90
15
30
90
B90
150
30
25
20
15
Unfiltered
Unfiltered
Unfiltered
15
60
229
234
233
249
270
258
265
244
5.5
20.3
13.6
8.6
7.0
5.7
24.6
15.9
7.3
Flood Milled
5
15
30
60
A90
15
30
90
B90
180
30
25
20
15
Unfiltered
Unfiltered
Unfiltered
15
56
142
153
148
149
167
170
175
146
13.1
2.3
5.8
4.5
5.2
6.4
6.5
3.5
13.6
Row 40 Brown
5
15
30
60
A90
15
30
90
B90
180
30
25
20
15
Unfiltered
Unfiltered
Unfiltered
15
3
20
23
22
25
26
30
31
27
1.0
2.2
2.2
2.0
2.6
5.8
1.2
1.5
1.2
Row 40 Milled
5
15
30
60
A90
15
30
90
B90
180
30
25
20
15
Unfiltered
Unfiltered
Unfiltered
15
8
21
21
22
22
25
25
25
21
0.9
1.6
0.8
1.6
0.6
1.0
1.9
1.3
1.4
15
30
15
30
30
25
Unfiltered
Unfiltered
81.3
85.1
90.6
92.9
6.6
1.9
2.0
1.8
NIST 1568B
Certified =
92±10
18
Possible Approach for iAs analysis of truckloads of rice arriving at rice mills:
A method to analyze samples of truckloads before delivery at a rice mill may not be
necessary with the present CODEX limit of 0.20 mg iAs/kg fresh weight of milled rice because
most US rice meets the CODEX iAs limit. Some rice grown with continuous flood on As
enriched soils does exceed the CODEX limit, but mixtures from many producers and fields are
very unlikely to exceed the iAs limit based on US-FDA sampling and analysis of market
samples of rice in the US. In addition, rice mills have worked to identify producers who use
AWD or otherwise can supply rice which is well below the US-FDA guidance for iAs in infant
rice cereals (0.10 mg iAs/kg fresh weight), and satisfy the regulatory limits for the small market
for infant cereal by purchase of low iAs rice from these carefully managed sources. Thus a
method to sample and analyze iAs in milled rice from arriving trucks may not be needed by the
rice industry at this time.
Although present truckload sampling and evaluation methods of the rice industry would not
easily include extraction and analysis of iAs in truckloads of rice as they arrive at the mills, one
can construct such a possible method, if it were needed, based on our experience. Trucks
arrive at the mills and remain there for many hours before delivering their loads (based on
image of rice mill with over two hundred large trucks awaiting delivery). The steps would
include:
1. Representative sampling of truckloads (as presently conducted at time of delivery).
2. Mechanical husking a portion of the sample.
3. Rapidly microwave oven drying of a sample of husked (brown) rice.
4. Milling the sample of husked rice.
5. Grinding the milled rice to a fine powder using simple coffee grinder.
6. Extraction of iAs (30 min. HotBlock extraction with 0.28 M HNO3 method reported
above).
7. Filtering the extracted sample (30 min).
8. Pre-reduction with 30 min. incubation.
9. Analysis of iAs using HG-ICP-AES or other appropriate method (2 min./sample).
A rapid milling process would be difficult to achieve because moisture content affects
milling yield. But broken kernels could be analyzed with unbroken milled kernels if necessary.
We believe iAs analysis of truckloads of rough rice could be accomplished while trucks were on
line to deliver their loads so that high iAs loads could be segregated from low iAs loads if this
became necessary for the industry.
19
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Appendix 1. Standard Operating for Extraction of As from Powdered Rice following US-FDA methodology.
Appendix 2. Standard Operating Procedure for Hydride Generation analysis of iAs in extracts of rice.
22
HotBlock Standard Operating Procedure for Inorganic Arsenic in Rice Grain (Jan. 8, 2016)
1. Rice samples must be ground before extraction using the HotBlock (SCP Science DigiPrep MS). Refer to
the standard operating procedure for use of UDY Cyclone Sample Mill (Catalog # 3010-030) for grinding
rice (using 0.8 mm stainless steel screen).
2. If not already in a hood, transport the HotBlock into a hood. Set the HotBlock to preheat to 95°C before
weighing samples. This way the HotBlock will be preheated and ready for use after samples are
prepared. Ensure that there is about 20 mL of 0.28 M HNO3 in the temperature probe tube for accurate
temperature reading. *NOTE: You must change out the 0.28 M HNO3 solution in the temperature probe
tube every day. If you do not, over time the water will evaporate and you will have an increasing
amount of HNO3 in the tube. If the tube looks discolored or cracked over time, replace it with a new
one. In each run of the HotBlock, include 2 standard rice samples and 4 blanks; include 1 duplicate per
10 samples.
3. Rice samples are weighed into 50 mL DigiTUBES (SCP Science 010-500-263) (Note: Tubes were checked
for metal contamination and do not need to be acid washed first). Number each tube and weigh 0.700 g
of rice sample into the tube. Record the tube number, sample description and weight in record book.
Cover the tube with a blue lid but do not screw lid on tightly yet. The HotBlock can digest 47 samples in
one run; you may use blue trays to hold tubes for easy transporting.
4. Once you have weighed out all samples, add 10 mL of 0.28 M HNO3 to each sample tube using a
repipettor and screw the blue lid on tightly. Vortex all samples to mix well; assure rice powder is
suspended by checking bottom of tube.
5. Check that the HotBlock has preheated to 95°C. Load the samples into the HotBlock and start a method
with parameters to heat to 95°C and to hold for 90 minutes (standard FDA extraction method).
6. While the rice is digesting, set up for filtration. Number new 50 mL DigiTUBES and place under filtration
racks. Use polyethylene funnels and Whatman #40 filter paper. Filter paper should be wetted with 0.28
M HNO3 before filtration and the rinse discarded to the proper hazardous waste container.
7. Once HotBlock is finished the program, immediately transfer tubes into blue racks to cool. Once cool to
the touch, pour each sample from tube into filter paper; then rinse (limit volume used) the tube again
into the filter with 0.28 M HNO3. After this has drained, rinse the filter paper (limit volume used) well
starting at the top with 0.28 M HNO3. Bring to 20 mL volume with 0.28 M HNO3 and cap with a new lid.
Invert capped tube to mix sample. Dispose of original digestion tubes and lids.
8. Refer to the standard operating procedure for hydride generation for arsenic analysis for instruction on
how to run the samples on the ICP for inorganic arsenic. Ensure that you follow the instructions for
adding antifoam to the SB solution and that you use 4 M HCl KI/AA. Note that this is different from
running total arsenic samples.
Hydride Generation for Inorganic Arsenic Analysis Standard Operating Procedure
Jan. 8, 2016
NOTE: This SOP is used for rice samples that were extracted for iAs using the Hotblock Procedure.
Basis for Analysis:
An acidified sample/blank/standard goes through a pre-reduction step utilizing a potassium
iodide/ascorbic acid mixture as the reducing agent for As (assuring the analyte is in the lower oxidation
state - As5+ to As3+). This prereduced sample is then mixed with a pumped stream of reductant (sodium
borohydride stabilized with sodium hydroxide), to produce the gaseous hydride (equation below). At
the point of reaction, hydrogen gas is produced as a by-product, resulting in a two phase mixture. A
flow of argon is added to this mixture and the hydrides are “stripped” into the gas phase. A gas/liquid
separator allows the gaseous, hydride containing phase to enter the ICP for analysis, and allows the
remaining liquid to be pumped to waste. The ICP spectrometer’s mass-flow controlled “nebulizer argon”
is used as the source of the hydride stripping argon. An example of hydride generation reaction is:
NaBH4 + 3H2O + HCl → H3BO3 + NaCl + 8H + E → EHn + H2
(Where E is the analyte element)
Reagents:
0.5% Sodium Borohydride in 0.05% Sodium Hydroxide (SB) – prepared daily
Weigh 0.5 g sodium hydroxide into 1 L volumetric flask. Add about 100 mL DI water to dissolve.
Weigh 5.0 g sodium borohydride and add to the volumetric flask. Bring to volume with DI water
and mix to dissolve with stir bar. The ICP uses approximately 100 mL of solution/10 samples or
2.5 mL of solution/min. One liter of solution is sufficient for processing ~100 samples, or about
5-6 hours of continuous use of the ICP-OES. Make up more solution as needed. (If running fewer
samples make 500 mL instead)
Volume
1L
500 mL
200 mL
NaOH
0.5 g
0.25 g
0.1 g
SB
5.0 g
2.5 g
1.0 g
This solution must be filtered before use, with Whatman “3” Filter Papers (Whatman no.
1003-150) 150 mm diameter.
After filtration add 0.9 mL Antifoam B Emulsion (Sigma A5757-250ML) for every 250 mL of
sodium borohydride/NaOH solution. IMPORTANT NOTE: You must add antifoam solution to
SB/NaOH solution or intense bubbling will cause the plasma to extinguish. If you use antifoam in
the SB the hydride setup must be taken apart and flushed with DI H20 before you leave for the
night or it will leave dried up residue inside of the bubbler and tubing.
4 M HCl 5% Potassium Iodide and 5% Ascorbic Acid (KI/AA) – prepared daily
Weigh 25 g potassium iodide and 25 g ascorbic acid into 500 mL volumetric flask. Add 164 mL
concentrated HCl to the volumetric to make the solution 4 M HCl. Bring to volume with DI
water and mix to dissolve with stir bar.
1
Volume
500 mL
250 mL
200 mL
100 mL
KI
25 g
12.5 g
10.0 g
5.0 g
AA
25 g
12.5 g
10.0 g
5.0 g
HCl
164 mL
82 mL
65.6 mL
32.8 mL
1.73 mol/L sulfamic acid (SA)
Weigh 16.78 g sulfamic acid into 100 mL volumetric flask. Bring to volume with DI water and mix
to dissolve with stir bar. Transfer to a labeled plastic bottle for future use. This solution does not
have to be prepared daily.
Reagent Notes: When starting the day, prepare the SB first as it needs to be filtered before use. The
KI/AA solution also takes a little longer to fully dissolve; in the meanwhile, you can set up the filters for
the SB. Both KI/AA and SB take no more than a few minutes to dissolve. Sulfamic acid (SA) will take
longer to dissolve (up to 15-20 minutes) so if you know you are low on SA, prepare as early as possible
to avoid holding up sample preparation.
Order of preparation for a run: Make up reagents; Make up the standards; while these are sitting for
20 minutes, one can start preparing the samples. By the time samples are made the standards will
have sat the needed 20 minutes and the ICP check/run can begin.
Sample Preparation:
4.0 mL digested sample (hot block sample)
1.5 mL concentrated HCl
3.0 mL HCl/KI/AA
2.5 mL sulfamic acid
= 11 mL total
When computing solution ppb from ICP results – this is a 2.75 dilution factor
Always add sulfamic acid last, and do so slowly to prevent violent bubbling and loss of sample. Also be
sure to do this in a hood. Bubbling, discoloration, and even precipitation can occur in random samples,
but more often than not occur in “blank” samples where the concentration of nitric acid may be higher.
Shake with vortexer after gases dissipate and allow to stand for 20 minutes for reduction reaction to
occur. Samples cannot be kept for more than one day.
Samples take about 2 min. to run – For total run time, need to figure stds/QCs/ and spikes in addition to
samples, in order to figure out how many you can run in a day. Typically with the addition of stds/QCs a
set of 30 samples takes about 2 hours to run through from start to finish. Generally spikes every 10
samples.
Standards
Prepare As Stock solutions used to make the standards.
For 1 ppm Stock: Pipette 0.1 mL 1000 ppm stock As solution into 100 mL volumetric flask. Bring
to volume using 1 N Trace Element Grade HCl and mix well. Make new batch every month to
ensure the accuracy of the standard.
For 10 ppm Stock: Pipette 1.0 mL 1000 ppm stock As solution into 100 mL volumetric flask. Bring
to volume using 1 N Trace Element Grade HCl and mix well. This solution will also be used for
spikes.
2
Prepare standards in volumetric flasks. Example concentrations of calibration and QC standards are
below. Then add the reagents and lastly, bring to volume with 1 N Trace Element Grade HCl. Keep in
mind these standards have to be made daily and should not be saved past one day of use. Allow to sit
for 20 minutes before using.
Always prepare a 2.5 ppb standard daily for a start up check. Intensity usually hovers between 10001300 counts, but will occasionally be as high as 1500 counts. (ICP is set on Auto Read Parameters with a
minimum of 5 seconds and a maximum of 20 seconds per replicate).
When calibration is complete, except for the blank, RSD’s should be below 5.0 and should have at least a
0.999 Correlation Coefficient for the calibration curve. If not, correct the problem before starting to run
samples. QC’s should ideally be within 10% for the primary wavelength As 189. Seek assistance if
problems continue.
Hydride
Std
CONC
Vol
1.0 ppm CONC
Vol
1.0 ppm CONC
Vol
10.0 ppm CONC
Vol
10.0 ppm CONC
Size
As Stock Std
Size
As Stock Std
Size
As Stock Std
Size
As Stock Std
As, ppb
mL
mL
mL
mL
mL
mL
mL
mL
Blank
50
0
100
0
-
-
-
-
0.25
-
-
100
0.025
-
-
-
-
0.5
50
0.025
100
0.05
-
-
-
-
1.0
50
0.050
100
0.10
-
-
-
-
2.5
50
0.125
100
0.25
-
-
-
-
5.0
50
0.250
100
0.50
-
-
-
-
10.0
50
0.500
100
1.00
-
-
-
-
20.0
50
1.000
100
2.00
-
-
-
-
25.0
50
1.25
100
2.5
-
-
100
0.25
40.0
50
2.00
100
4.0
50
0.2
100
0.4
50.0
50
2.50
100
5.0
50
0.25
100
0.5
75.0
50
3.75
100
7.5
50
-
100
0.75
100.0
50
5.00
100
10.0
50
0.5
100
1.0
Total Volume
KI/AA
(mL)
Conc. HCl (mL)
25mL
6
3
50mL
12
6
100mL
24
12
.
3
Sample calibration range and approximate corrected intensity counts:
Standard
Intensity Counts (As 188.9 primary
wavelength)
Blank
-6.6 - 100
1.00 ppb
500-600
2.50 ppb
1100-1300 (as high as 1500)
5.00 ppb
2500- 3800
10.0 ppb
5000 - 7200
20.0 ppb
11000-13000
Spikes
Spike concentrations were determined by multiplying average sample readings by 3x. For example,
typical plant samples were spiked at 10 ppb and 20 ppb alternating every 10 samples. Soil samples were
spiked at 10 ppb and 40 ppb as As levels tended to be higher.
For best results, make sure samples that are blanks, NISTs, and other standards are not spiked.
Samples were spiked using a 10.0 ppm As stock solution (as prepared above).
Spike level (concentration)
Spike volume (of 10 ppm stock) in 11 mL sample
10 ppb
20 ppb
40 ppb
11 µL
22 µL
44 µL
In general, spike recovery for As is very good. All but one or two spike sets come back within 90% (most
closer to 100%) recovery in a set of 300 samples. In smaller sets of samples, we were able to have close
to 100% recovery for all spikes.
Instrument Parameters
PerkinElmer Optima DV 4300 is operated in axial mode, using a Glass Liquid Separator set-up.
Using the recommended tubing and probe, the set-up uses approx. 5 mL/min. of sample and 2.5
mL/min. of sodium borohydride.
Instrument Settings:
Rinse time
Plasma Gas
Auxilary Gas
Nebulizer Gas
Flow rate
Power
Torch
Readings
Read Parameters
Wavelengths
When autosampler is in location, plus 30 seconds
16 L/min
0.2 L/min
0.60 L/min
1.5m L/min
1450 watts
-2.0
Peak Area, 3 points per peak, 2 pt. background
3 reps
min 5 s integration time
max 20 s integration time
As188.9 primary, As193 secondary check
4
Purge gas
Axial
Read Delay
nitrogen
Viewing Distance 15.0
40 s
Autosampler Probe Rinse Time:
Due to the larger sampler tubing, the rinse time needs to be extended for the autosampler. An
additional 30 sec. works well to insure there is enough rinse in the rinse port. (Can be changed by
going into Options/autosampler).
Clean-up
Samples cannot be kept overnight, so only prepare as much as you can run on the same day. Dump
samples in the appropriate acid waste container and fill empty tubes from the analysis run with soapy
water until they are taken to an acid bath. When dumping SB and KI/AA into the acid waste container,
some foaming is common.
If running again the next day glassware can reused if rinsed with water (beakers containing SB and
KI/AA, funnels for filtering SB, volumetrics used for making reagents) or 1 N HCl (volumetric flasks used
for making standards, including the stoppers). If you will not run hydride again for several days or weeks,
then all glassware should be submerged in soapy water and cleaned in an acid bath.
When no further As analyses are scheduled, volumetric flasks and plastic standards tubes should be
rinsed and put into the appropriate holding bucket used for standard equipment until they are cleaned
in an acid bath.
The bubbler, tubing and mixing block must be flushed with DI H20 at the end of the day to rinse it of
the antifoam residue. If you do not perform this step the residue can dry inside of the hydride setup
and cause problems.
Set-up:
NOTE: Tubing should be changed weekly with heavy (daily) use or more frequently as needed.
Flow Apparatus:
Different fittings can be used, but our current set-up is shown (see photos) and parts are listed in the
table below. A set of two mixing blocks are used to combine the sample and reductant streams and then
to add the stripping argon. The gaseous hydrides are transported by the stripping argon flow directly
into the base of the Option 4300DV.
Photo Label
PE Part #
QTY
Liquid separator (Comes with fittings)
B0193772
1
B
tubing with sinker
B0191059
1
C
blue to blue - 1.0 mm dia, 110 mm PTFE tubing.
B0191058
2
D
blue to blue 1.0 mm dia, 300 mm PTFE tubing.
B0198097
2
E
adapter E
B0196857
1
F
adapter A
B0193342
2
G
adapter C
B0196850
2
A
Item #
5
H
adapter K
B0507918
1
I
adapter L
B0507920
1
J
mixing block
B0507962
2
K
silicon tubing
B0018283
1
L
autosampler probe
B3000055
1
M
sample tube
B191060
1
N
injector adaptor
N069-5426
1
O
Connector 1B
B0196882
1
P
1.14 mm peristaltic pump tubing, red/red, Reductant
Q
1.52 mm peristaltic pump tubing, blue/yellow, Sample
Rainin 39-625
R
3.18 mm peristaltic pump tubing, black/white, Waste
Rainin 39-628
S
PVC Tubing 3 mm i.d.
Fisher Brand 14-190524
B0048139
6
7
Sources
“Continuous Flow Hydride Generation using the Optima4300DV ICP,” Bosnak CP and Davidowski L
(2004)
Damkroger G, Grote M, Jansen E (1997) Fresenius J of Anal Chem 357: 817-821
8