Robert Scott and Joseph Granchelli

Key Words
Bottles, HDPE, metal content

GoalHighlighting the extremely low content of metals in Thermo Scientific™
Nalgene™ plastic bottles.

Abstract
In many systems plastic bottles are used to contain reagents, intermediates,and products in solution. In such systems, it is critical to ensure that thebottle remains strictly a container and does not interact with the contents
to change them in any way. Low levels of certain metals are often foundin plastic resins, and due to the nature of plastic these can be extracted
from the resin by many solvents. Here, we examine the metal content ofhigh density polyethylene (HDPE) bottles from several different brands, andfind that Thermo Scientific Nalgene HDPE bottles contain lower levels ofmetals than competitors. These bottles have a clear advantage for use incontaining reagents that are sensitive to contamination from metals.

Introduction
Containers made of plastic resins are used to contain awide variety of materials in many different settings.
Especially in the biological and chemical fields, thesematerials can include high-value reagents, intermediates,or products; or critical components in sensitive assays.
Some of these types of materials can be extremely sensitive
to reactive compounds that may be found in the plastic,even at low concentrations. In such cases, the value of thecontainer goes beyond the ability to maintain the closure
seal and includes any manner in which the plastic
container itself alters the contents. While plastic
containers are a generally inexpensive solution for storageof reagents or products, low-quality containers may resultin higher costs in the long run through the contaminationand subsequent loss of high-value contents. Part of thequality offering on these types of containers is theverification that they do not leach detrimental substances
into the solutions contained within. Metal contamination
is of particular interest in plastic containers, since they areoften present at some levels in the plastic base resin andcan react readily with many solutions that are normallystored in them. Here, we examined the metal content, as
well as limited other contaminants, of several different
bottles made with a high-density polyethylene (HDPE)
base resin, and compared bottles from Thermo ScientificNalgene to several competitors.

Appli cat i on N ote

Analysis of metals content in
Thermo Scientific™ Nalgene™ HDPE bottles
and competitors

2Experimental details:
Metals were evaluated by High Resolution Inductively
Coupled Plasma Mass Spectrometery (ICP-MS).
Sample Preparation
Three bottles of each brand were tested. A portion(approx. 200-250 mg) of plastic was cut from the body ofeach bottle using a zirconium ceramic-blade knife thatwas cleaned between each sample. Each plastic piece wasdeposited directly into a polypropylene centrifuge tube forcleaning. The goal of cleaning was to remove any surface
contamination resulting from the cutting process.
Approximately 2 mL of a 2% solution of nitric acid
(HNO3, Fisher brand Optima grade) in ultrapure(18.2 MΩ-cm) water was added to each tube. The tube
was shaken for about one minute, and then the 2% HNO3
was poured off of the sample. Ultrapure water was used
to rinse the sample and tube twice, and the sample wasallowed to dry at room temperature inside the tube.
Sample Digestion
Cleaned, dried samples were weighed into Teflon digestion
vessels and nitric acid (3.5 mL) and Fluka brandTraceSelect Ultra grade 30% hydrogen peroxide solution(H2O2, 1.5 mL) were added to each vessel. The vessels
were sealed and heated to 190°C in a microwave digester.
After the vessels cooled, they were opened and thecontents rinsed into a pre-cleaned, pre-weighed 2 oz.
Nalgene LDPE bottle. The digested samples (“digestates”)
were diluted to approximately 50 g total with ultrapure
water. Two samples of Nalgene LDPE bottles were also
digested for comparison. Spike recovery tests wereperformed for additional portions of three plastic bottles.
A known amount of each element of interest (except Hg)
in the form of a liquid standard was added to the sample
prior to digestion. Results for unspiked and spiked
replicates were compared in order to calculate percent
recoveries. These recoveries are listed in the results table.

1000

Vessel blanks (empty vessels microwaved with digestion
reagents) were prepared and analyzed alongside thesamples in order to check for vessel and/or reagent
backgrounds. These backgrounds were found to beinsignificant.
Preparation of standards: Instrument calibration
standards were prepared from commercially purchased(High Purity Standards) single- and multi- elemental
solutions. Standards were prepared gravimetrically, withweighing at every step, in order to accurately calculate
exact concentration values. 2% HNO3 was used as adiluent. The standards consisted of four series, each withfive different concentrations, which encompassed allelements of interest (except for Hg). Digested polymer
samples were diluted for analysis by a factor of 2 using
ultrapure water. For those samples that contained very
high levels of certain elements, further dilutions andre-analyses were performed. Internal standards Sc, In, andTl were added to all samples and standards. Internalstandards are used to correct for matrix effects in theinstrument.
Instrumental Analysis: All samples were analyzed using a
VG Axiom high-resolution ICP-MS. A total of four
instrumental runs were required to analyze all samples.
For each run, the necessary standards were analyzed tocalibrate the ICP-MS. Blanks and QC standards wereanalyzed throughout each run in order to monitor
backgrounds and ensure continuous and correct
instrument response. The calibration for Hg wascalculated based on the response of Tl and a measured
historical response factor. Thus the Hg limits of detection
should be regarded as semi-quantitative. No Hg wasdetected in any of the samples. For the polymer samples,
results are reported in units of ppb, or parts per billion byweight (ng/g), relative to the solid plastic.

HDPE Bottle Metal Content (ppm)
776

100

116

144

167

62

101

1

NalgeneBrand A

Brand B

Brand C

Brand D

Brand E

Figure 1. Total metal content (plus silicon and phosphorous) in various brands of HDPE bottle, in parts per million (ppm). Y-axis islogarithmic.

Results and discussion

3250 and 500 mL plastic bottles were analyzed for content
of selected metals as well as silicon, phosphorous, andantimony. Nalgene bottles were found to contain far lesstotal metal contamination than nearest tested competitor.
With silicon and phosphorous included, the nearest
competitor was more than an order of magnitude higher
in contamination than Nalgene bottles (Figure 1). On
examination of individual contaminants (Table 1),
Nalgene bottles were found to have the lowest levels (orbelow the limit of detection) in 12 out of the 17
substances tested for, and were very near the lowest levels
in 4 of the remaining substances. Nalgene bottles werealso below the limit of detection for biologically toxic leadand mercury. In comparison, several competitors had
contamination levels several orders of magnitude higher
than Nalgene with some or many of the substances tested.
In some cases, these measurements ranged into the tens
(or hundreds) of parts per million, whereas contaminationin Nalgene bottles was found only in parts per billion.

inactivation. Bottles used for packaging peroxidase
reaction reagents are very sensitive to iron and manganeseas these can degrade hydrogen peroxide, reducing assay
sensitivity. Reagents used for testing blood levels of
calcium or magnesium are also sensitive to contaminationfrom these metals which may alter test results. The highquality of both the virgin plastic resin and the process andtooling used to mold these bottles allows Nalgene toproduce bottles with very low metal content. In suchhighly sensitive systems, using Nalgene bottles would bevery beneficial in maintaining the integrity of any assays
using reagents stored in plastic bottles.

Conclusion
The extremely low quantity of detectable metals isindicative of the high quality of both the resin and theprocess used to manufacture Nalgene HDPE bottles.

It is important to note that due to the digestion processused for this study, the levels found are higher than thoselikely to be found in the bottle contents. This test
represents the worst-case scenario of all metals that could
possibly be extracted from the plastic. However, knowing
the levels of contaminants is important, especially sincecontainers for diagnostics and specialty packaging often
are used for intermediate storage of reagents used inhighly sensitive analytical chemistry and critical assays. In
such applications, even small concentrations of specificmetals can alter results. For example calcium, magnesium,and zinc can cause protein precipitation and/or enzyme

Nalgene,
500 mL HDPE

Brand A,
500 mL HDPE

Brand B,
500 mL HDPE

Brand C,
500 mL HDPE

Brand D,
250 mL HDPE

Brand E,
500 mL HDPENa<LOD

<LOD

393

218

<LOD167Mg

<LOD

2637

8,454

740

109

Al

383

311

2,702

9,457

12,949

392

Si

147

46,136

96,05010,050267

137,981P

152

13,285

15,377

20,251

88142

Ca

<LOD

164

85

93,308

122,537

423

Ti

466

1,177

15

1,544

29,447

198

Cr

<LOD

770

1,229

3

117

1,991

Fe

68

55

45258

513

235

Ni

<LOD

<LOD

<LOD

<LOD

70111

Zn

<LOD

22163

110

249

634,080

Mo

<LOD

<LOD

6<LOD

9<LOD

Cd

3

<LOD

4<LOD

<LOD

<LOD

Sn

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

Sb

<LOD

<LOD

<LOD

<LOD

7<LOD

Hg

<LOD

<LOD

<LOD

<LOD

<LOD

<LOD

Pb

<LOD

<LOD

<LOD

168Total

1,220

61,946116,107

143,652

167,000

775,836

Table 1. Levels in parts per billion (ppb) of substances extracted from samples taken from several HDPE bottles. <LOD indicates thesample contained below the level of detection for the indicated substance.

Appli cat i on N ote

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