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Open-source autosampler for elemental and isotopic analyses of solids
Matheus C. Carvalho, William Eickhoff, Michael Drexl
PII: S2468-0672(20)30032-8
DOI: https://doi.org/10.1016/j.ohx.2020.e00123
Reference: OHX 123
To appear in: HardwareX
Received Date: 25 July 2019
Revised Date: 25 June 2020
Accepted Date: 6 July 2020
Please cite this article as: M.C. Carvalho, W. Eickhoff, M. Drexl, Open-source autosampler for elemental and
isotopic analyses of solids, HardwareX (2020), doi: https://doi.org/10.1016/j.ohx.2020.e00123
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover
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© 2020 Published by Elsevier Ltd.
Title: Open-source autosampler for elemental and isotopic analyses of solids
Authors: Matheus C. Carvalho1, William Eickhoff2, Michael Drexl3
Affiliations: 1Centre for Coastal Biogeochemistry Research. School of Environment, Science
and Engineering. Southern Cross University. PO box 157, Lismore, 2480, NSW, Australia.
2 Southern Cross Plant Science. Southern Cross University. PO box 157, Lismore, 2480, NSW,
Australia.
3 Southern Cross Geoscience. Southern Cross University. PO box 157, Lismore, 2480, NSW,
Australia.
Contact email: mcarvalh@scu.edu.au
Abstract: Elemental and isotopic analyses are performed using elemental analyzers, and are
widely employed for diverse scientific fields. An elemental analyzer is typically equipped with
an autosampler. Here we present an open-source autosampler for elemental and isotopic analysis
of solid samples. The autosampler consists of 1) a sampling table, on which a carousel pushes
samples inside an orifice, and 2) a purging pipe, placed directly beneath the orifice, where the
sample is purged off surrounding air, and then delivered to the reaction tube. The action of the
purging pipe ensured that air contamination, an issue for the analysis of some elements like
nitrogen and oxygen, was negligible, and results for elemental and isotopic composition of
nitrogen and carbon were inside specs. Compared to commercial alternatives, the autosampler
presented here has the advantages of lower cost to build and maintain, universal compatibility
with instruments from different manufacturers, capacity do deal with bulky samples, capacity for
a larger number of samples in a single run, and no necessity for time-consuming purging in-
between sample loading. The autosampler could potentially also be employed for the analyses of
other elements (e.g. oxygen, hydrogen and sulfur) because they are performed using similar
equipment.
Keywords: 3D printing; Autosampler; Arduino; AutoIt; carbon; elemental analysis; laboratory
automation; nitrogen; OpenSCAD; stable isotopes; sampling
Specifications table
Hardware name
Oseas
Subject area
Chemistry and Biochemistry
Environmental, Planetary and Agricultural Sciences
Medicine, Forensics
Hardware type
Solid sample handling
Open Source License
GNU General Public License (GPL) 3.0
Cost of Hardware
AU$ 542 plus extras
Source File Repository
https://osf.io/utzse/ or https://doi.org/10.17605/OSF.IO/UTZSE
1. Hardware in context
Elemental analysis is one of the most fundamental chemical measurements. Of special
significance for environmental and ecological research are the analyses of carbon and nitrogen
amounts and isotopic compositions in bulk samples, which are carried using elemental analyzers
operating on variations of the Dumas principle [1,2]. These analyzes used to be laborious and
time consuming, but became very popular after automation, which allowed rapid throughput and
easiness of operation [3,4,2]. Since then, these measurements have been performed in numerous
environmental and geochemical studies covering all sorts of environments [5-7]. They have also
been employed for forensic investigations, including drug testing, food provenancing, and crime
solving [8].
Elemental analyses of carbon and nitrogen are carried out using elemental analyzers.
Autosamplers are a standard part of most elemental analyzers, and some accessories for these
autosamplers have been developed by scientists, either to deal with special analytical needs [9],
or to make operation easier [10]. Autosamplers themselves are also among the most accessible
scientific devices in terms of fabrication by non-specialized engineers, with several examples
available in the literature [11-20]. While in some cases these autosamplers have been integrated
to other in-house made components [17,16,13,18,19] , the integration of in-house made
autosamplers to commercial, off-the-shelf equipment like elemental analyzers has been a
difficult challenge for several decades [21-23], only recently facilitated by the adoption of AutoIt
for laboratory automation.
AutoIt is a scripting language for the Windows operating system that allows easy
integration of analytical devices [24-27]. By means of AutoIt, open-source autosamplers for a
range of analytical purposes have been presented, covering both fluid (liquids and gases) [28,29]
and solid samples [30]. However, none of these autosamplers is appropriate to handle solid
samples for elemental analysis, because in these devices the samples must be introduced into a
reactor without air contamination. Here we present an open-source autosampler with the
necessary characteristics for elemental analysis that can fully replace commercial models for a
comparatively small cost (AU$540 plus extras).
2. Hardware description
The autosampler described here consists of two parts: 1) a sampling table and 2) a purging
pipe (Fig. 1). The sampling table consists of a table on which a carousel is placed (Fig. 2). The
carousel contains five concentric circles of holes. There are two stepper motors: one spins the
carousel, while the other pushes a gantry holding the carousel forward so that the inner circles
containing samples can also spin over the orifice, delivering the samples.
Fig. 1: EA autosampler mounted on top of an elemental analyzer. The sample table is the
four-legged acrylic sheet supporting the carousel. The purging pipe is the pipe beneath the
sampling table and connected to the elemental analyzer.
The purging pipe consists of 3 ball valves connected in series, each controlled by a servo
motor (Fig. 2). All valves and connectors in the setup are half inch in diameter, except those
from the pipe to helium supply, which need to be reduced to 1/8 or 1/16 inch diameters,
depending on the instrument’s specifications. The topmost part of the pipe is simply a straight
pipe or connector to minimize the space between the orifice of the plate holding the carousel and
the topmost valve. Just beneath are the top and middle valves, which are interconnected using a
simple connector. The space between the two valves is called “purging chamber”. The middle
valve is connected to the bottom valve using a T piece, which is connected to a carrier gas line.
The space between the middle and bottom valve is called “isolation chamber”. The bottom valve
is connected to the elemental analyzer using another T piece, which is also connected to a carrier
gas supply.
Fig. 2: EA autosampler. A table made of aluminum extrusion profiles holds an acrylic
sheet, on top of which a carousel spins delivering samples through a hole on the sheet. The hole
is placed directly above the purging pipe, where three ball valves, actuated by servo motors,
ensure that minimal air contamination occurs when the sample is delivered to the elemental
analyzer.
The purging pipe ensures that a minimal amount of air is carried along the sample for
analysis (Fig. 3). The sample falls on top of the top valve, which is opened and partially closed
after the sample drops through it. Then the middle valve is partially opened, such that carrier gas
purges the purging chamber. After a certain time (typically 1 min), the top valve is closed, and
the middle valve is fully opened, so that the sample falls on top of the bottom valve, inside the
isolation chamber. The middle valve is fully closed, and then the bottom valve is fully opened,
allowing the sample inside the elemental analyzer. The bottom valve is then immediately fully
closed, thus minimizing air contamination.
Fig. 3: Scheme of the operation of the purging pipe. Arrows indicate helium flow. Full
description in the text, section 2.
Both stepper and servo motors are controlled by a MKS Gen-L control board, also used in
other autosamplers [29,30]. The firmware of the board is Marlin, fully open-source, enabling the
use of G-code [29,30]. The integration of the autosampler and elemental analyzer is done using
AutoIt [28-30,24].
A video showing the autosampler working is presented in Supplementary Information 1.
3. Design files
3.3 Design Files Summary
In addition to the hyperlinks provided for the location of the files, this DOI link is also
available for the master page with all links: https://doi.org/10.17605/OSF.IO/UTZSE.
Design file
name
File type
Open source license
Location of the file
Carousel
OpenSCAD
GNU General Public
License (GPL) 3.0
https://osf.io/vs9py/
Gantry
support
Libre Office
Draw
GNU General Public
License (GPL) 3.0
https://osf.io/ezgcv/
Table top
Libre Office
Draw
GNU General Public
License (GPL) 3.0
https://osf.io/ezgcv/
NEMA 17
stepper
motor mount
OpenSCAD
GNU General Public
License (GPL) 3.0
https://osf.io/d64x5/
Servo to
ball-valve
connector
OpenSCAD
GNU General Public
License (GPL) 3.0
https://osf.io/gwxkr/
Spacer for 4
mm screws
OpenSCAD
GNU General Public
License (GPL) 3.0
https://osf.io/mv5ju/
Spacer for 3
mm screws
OpenSCAD
GNU General Public
License (GPL) 3.0
https://osf.io/4kmwf/
Sieve for
half inch
pipe
OpenSCAD
GNU General Public
License (GPL) 3.0
https://osf.io/qjpfu/
4. Bill of Materials
Freight costs are not included in this table. They can be significant for bulky items, like extrusion
profiles. Also, specific connections between the purging pipe and the elemental analyzer are not
included, as they are likely to vary depending on the elemental analyzer model.
Figure
or
referen
ce
Compon
ent
Numb
er
Cost
per
unit
–
AU$
Total
cost –
AU$
Source of materials
Material
type
Fig. 2
T slot
20x20m
m, 35
cm
4
$3.8
5
$14.0
0
https://www.ebay.com.au/itm/2020-
Aluminium-Extrusion-T-Slot-Profile-Euro-
Standard/283479874340
Alumin
um
Figs 2
and 9
T slot
20x20m
m, 22
cm
7
$2.4
2
$16.9
4
https://www.ebay.com.au/itm/2020-
Aluminium-Extrusion-T-Slot-Profile-Euro-
Standard/283479874340
Alumin
um
Figs. 2
and 12
T slot
20x20m
m, 10
cm
3
$1.1
0
$3.30
https://www.ebay.com.au/itm/2020-
Aluminium-Extrusion-T-Slot-Profile-Euro-
Standard/283479874340
Alumin
um
Figs. 2
and 12
T slot
20x20m
m, 25
cm
1
$2.7
5
$2.75
https://www.ebay.com.au/itm/2020-
Aluminium-Extrusion-T-Slot-Profile-Euro-
Standard/283479874340
Alumin
um
Fig. 10
2028
bracket,
pack
with 20
1
$10.
15
$10.1
5
https://www.aliexpress.com/item/326427
11369.html
Alumin
um
Fig. 7
Long 90
degree
bracket
2
$1.0
6
$2.12
https://www.bunnings.com.au/carinya-
50-x-50-x-20-x-2mm-make-a-
bracket_p3960590
Steel
Figs.
2, 6
and 12
NEMA
17
stepper
motor
2
$13.
66
$27.3
2
https://www.ebay.com.au/itm/Nema-17-
Stepper-Motor-40Ncm-0-9A-4-Lead-
90cm-Lead-Cable-for-DIY-3D-Printer-
CNC/113127734830
Metal,
plastic
Fig. 13
Shaft
coupler,
5 mm to
1
$5.5
6
$5.56
https://www.ebay.com.au/itm/5-to-8mm-
Shaft-Coupler-Aluminium-Blue-
Coupling-CNC-Machine-Lead-Flux-
Workshop/113212021207
Metal
8 mm
Figs. 2
and 11
Acrylic
sheet,
A4 size
2
$9.5
0
$19.0
0
https://www.ebay.com.au/itm/Clear-
Acrylic-Precision-Cut-Sheets-
Commonly-known-as-perspex-
plexiglass/322038335759
Plastic
Figs. 6
and 19
M3
screws,
10 mm
length,
pack
with 10
2
$7.2
0
$14.4
0
https://www.ebay.com.au/itm/M3-M4-
M5-M6-M8-Socket-Head-Cap-Screw-
Stainless-Steel-304-Metric-
Coarse/142732934590
Metal
Fig. 15
M3
screws
(16 mm
length),
pack
with 10
1
$7.2
0
$7.20
https://www.ebay.com.au/itm/M3-M4-
M5-M6-M8-Socket-Head-Cap-Screw-
Stainless-Steel-304-Metric-
Coarse/142732934590
Metal
Fig. 15
M3 nuts,
pack
with 10
1
$5.5
0
$5.50
https://www.ebay.com.au/itm/263624205
539
Metal
Fig. 19
M3
hammer
nuts,
pack
with 10
1
$6.5
0
$6.50
https://www.ebay.com.au/itm/10-x-M3-
T-Slot-hammer-nuts-for-20-series-
extrusion-aluminium-
profile/113534394323
Metal
Fig. 16
M4
screws,
40 mm
length,
fully
threaded
, pack
with 10
1
$1.8
7
$1.87
https://www.aliexpress.com/item/330209
81797.html
Metal
Fig. 16
M4
hammer
nuts,
pack
with 10
1
$6.0
0
$6.00
https://www.ebay.com.au/itm/10-x-2020-
M4-T-Slot-hammer-nuts/113218156075
Metal
Fig. 16
M4 nuts,
pack
1
$5.5
0
$5.50
https://www.ebay.com.au/itm/263624205
539
Metal
with 10
Figs.
7, 8, 9,
10 and
12
M5
screws,
10 mm
length,
pack
with 100
1
$21.
00
$21.0
0
https://www.ebay.com.au/itm/M3-M4-
M5-M6-M8-Socket-Head-Cap-Screw-
Stainless-Steel-304-Metric-
Coarse/142732934590
Metal
Figs.
7, 8, 9,
10 and
12
Washer
for M5
screw,
pack
with 100
1
$9.8
0
$9.80
https://www.ebay.com.au/itm/142552908
688
Metal
Figs.
5, 7, 8,
9, 10
and 12
Hammer
nut for
M5
screw,
pack
with 100
2
$9.6
9
$19.3
8
https://www.ebay.com.au/itm/50x-
Hammer-Head-T-Nut-Drop-In-M5-for-
30-Series-European-Aluminum-
Slot/272532621625
Metal
Fig. 11
M5
screws
with
hammer-
head, 16
mm
length,
pack
with 10
1
$3.1
8
$3.18
https://www.ebay.com.au/itm/DIY-M5-
Hammer-Head-T-Bolt-Screw-For-
20Series-Aluminum-T-slot-10mm-
12mm16mm-25mm/312480457816
Metal
Fig. 11
Wingnut
for M5
screw,
pack
with 5
1
$8.9
0
$8.90
https://www.ebay.com.au/itm/263678710
533
Metal
Fig. 5
M5
screws,
35 mm
length,
fully
threaded
pack
with 10
1
$3.3
6
$3.36
https://www.aliexpress.com/item/328689
05756.html
Metal
Fig. 5
Spacer
for M5
screws,
10 mm
length,
set with
25
1
$2.4
7
$2.47
https://www.aliexpress.com/item/25pcs-
White-Plastic-Nylon-ABS-Round-Non-
Threaded-Column-Standoff-Support-Spacer-
Washer-For-M5-Screw/32650018699.html
Nylon
Figs. 6
and 9
GT2
Timing
belt, 1m
1
$1.8
3
$1.83
https://www.ebay.com.au/itm/1X-Courroie-
2GT-6mm-timing-belt-3D-printer-courroie-
GT2-1M-S3M8/233200843536
Rubber
Figs. 6
and 9
20 teeth
Pulley
for GT2
timing
belt, 20
teeth
1
$4.5
0
$4.50
https://www.ebay.com.au/itm/LearCNC-GT2-
Timing-Belt-Pulley-RepRap-Prusa-RAMPS-
Kossel-3D-Printer-Nema17/201145021195
Metal
Figs. 6
and 9
Wheels
625ZZ,
21.5 cm
in outer
diameter
, pack
with 9
1
$7.9
1
$7.91
https://www.aliexpress.com/item/9pcs-3D-
Printer-Kossel-Nylon-Plastic-Wheel-with-
Bearings-Bearing-Roller-Wheel-POM-5-x-
21/32816554334.html
Metal,
plastic
Fig. 4,
Sectio
n 3
3D-
printed
carousel
1
<$0.
10
<$0.1
0
Section 3.3
PLA
Fig.
16,
Sectio
n 3
3D-
printed
servo to
ball
valve
connecto
r
3
<$0.
10
<$0.3
0
Section 3.3
PLA
Fig.
16,
Sectio
n 3
3D-
printed
spacer
for M4
screws
6
<$0.
05
<$0.3
0
Section 3.3
PLA
Fig.
12,
Sectio
3D-
printed
NEMA
1
<$0.
10
<$0.1
0
Section 3.3
PLA
n 3
17 motor
mount
Fig.
18,
Sectio
n 3
3D-
printed
sieve for
half inch
pipe
1
<$0.
10
<$0.1
0
Section 3.3
PLA
Fig. 19
MKS
Gen L
control
board
with
stepper
motor
drivers
(A4988)
1
$32.
01
$32.0
1
https://www.ebay.com.au/itm/Part-A4988-
MKS-Gen-L-V1-0-Integrated-Mainboard-
Control-Board-Motherboard/123147319056
Metal,
plastic
Fig. 20
Cables
with
adapters
to
connect
stepper
motors
to
control
board
(1m), set
with 4
1
$3.7
5
$3.75
https://www.ebay.com.au/itm/4x-6-pin-
JST-to-4-pin-Dupont-Connector-for-
Nema17-Stepper-Motor-Cable-3D-
Printer/183199926610
Metal,
plastic
Fig. 20
Cable
wrapper
2m
1
$8.8
9
$8.89
https://www.ebay.com.au/itm/2M-Spiral-
Cable-Wrap-Tidy-Cord-Wire-Banding-
Storage-Organizer-10mm-25mm-3-
Colors/293034936624
Plastic
Fig. 20
USB
cable,
5m long
1
$6.5
0
$6.50
https://www.ebay.com.au/itm/USB-2-0-
Type-A-Male-to-B-Printer-Cable-for-
HP-Canon-Dell-Brother-Epson-
Xerox/111656844488
Metal,
plastic
Not
shown
24 V,
5.62A
power
supply
1
$29.
00
$29.0
0
https://www.ebay.com.au/itm/AU-Power-
Supply-Adapter-Transformer-AC240V-To-
DC24V-1-2-3-4-5A-for-LED-
Strip/202120736297
Metal,
plastic
Not
shown
Adapter
for
power
supply
to the
control
board
(2.1 mm
illuminat
ed
polarity
sensing
DC
socket)
1
$8
$8
https://www.ebay.com.au/itm/NEW-2-
1mm-Illuminated-Polarity-Sensing-DC-
Socket-WQ7289/252988850556
Metal,
plastic
Figs. 2
and 16
Hitec
HS-
805BB
servo
motor
3
$60.
00
$180.
00
https://www.modelflight.com.au/hitec-
hs-805bb-mega-quarter-scale-indirect-
drive-dual-ball-bearing.html
Metal,
plastic
Fig. 14
½ inch
ball
valve
3
$7.7
0
$22.1
0
https://www.bunnings.com.au/kinetic-
15mm-brass-ball-valve_p4790274
Brass
Fig. 18
½ inch
female-
female-
female T
tube
1
$4.5
0
$4.50
https://www.bunnings.com.au/garden-
rain-0-5-poly-irrigation-f-f-f-
tee_p3100169
Plastic
Fig. 18
½ inch
male-
male
connecto
r
4
$1.1
5
$4.60
https://www.bunnings.com.au/garden-
rain-0-5-poly-irrigation-
nipple_p3100149
Brass or
plastic
Fig. 2
½ inch
unthread
ed to
threaded
connecto
r
1
$0.9
5
$0.95
https://www.bunnings.com.au/holman-
15mm-x-1-2-pvc-valve-
socket_p3141883
PVC
Fig. 18
Thread
tape
(roll)
1
$3.0
0
$3.00
https://www.bunnings.com.au/gastite-
12mm-x-10m-yellow-
threadseal_p4920513
Teflon
Not
shown
O-ring
set
1
$3.8
5
$3.85
https://www.bunnings.com.au/kinetic-
assorted-sizes-o-ring-kit-43-
pack_p4920305
Rubber
Fig. 14
Perforate
d steel
stripping
(20 x 2 x
0.2 cm)
2
$1.7
2
$3.44
https://www.bunnings.com.au/carinya-
20-x-200-x-2mm-flat-make-a-
bracket_p3960600
Steel
5. Build Instructions
5.1: Custom parts
Acrylic sheets need be cut and drilled as per provided designs (Section 3).
3D-printed parts should be printed with at least 75% infill, except the carousel, for which 20%
suffices. It is recommended that the carousel is printed using clear plastic so that the holes can be
easily numbered using a pen or a marker (Fig. 4).
Fig. 4: Carousel made of clear PLA, allowing writing of the sample positions with a pen.
5.2: Assembling the sampling table
Fix the wheels to the acrylic supports (Fig. 5). First place a wheel (625ZZ, 21.5 cm in outer
diameter) around an M5, 35 mm screw, followed by a 10 mm nylon spacer. Attach the screw to
the acrylic plate and fix it using an M5 hammer nut. Do this for 4 screws for each of the two
plates.
Fig. 5. Wheels fixed to the one of the acrylic supports.
Fix one stepper motor to one of the acrylic supports using M3 screws (Fig. 6).
Fig. 6. Stepper motor fixed to one of the acrylic supports. Here only 3 screws are holding the
motor.
Fix long brackets to both acrylic supports using M5 screws, spacers and hammer nuts (Fig. 7).
Do not fix the brackets to the extrusion profile yet.
Fig. 7: Bracket fixed to acrylic support.
Slide each acrylic sheet already holding its wheels on a 22 cm t slot (Fig. 8).
Fig. 8: Acrylic support placed on a 22 cm aluminum extrusion profile. The support should move
smoothly with the wheels inside the groove.
Fix pulley on stepper motor, place timing belt around pulley, and fix it on t slot (Fig. 9). Use M5,
10 mm screws and hammer nuts to fix the belt to the profile. Align the pulley teeth to the groove
on the extrusion profile.
Fig. 9: Timing belt around stepper motor pulley, and fixed on aluminum extrusion profile.
Fix both 22 cm extrusion profiles to 35 cm ones, and so on for all the other profiles as in Fig. 2.
Connect the profiles using 2028 brackets, M5 screws, washers, and hammer nuts (Fig. 10).
Fig. 10: Bracket (2028) connecting extrusion profiles. Notice M5 screws and washers. Hammer
nuts are inside the extrusion profile grooves.
Fix 25 cm to both long brackets connected to acrylic supports (Figs 7 and 8).
Fix the acrylic table to the extrusion profiles using M5 hammer-head screws (the heads go inside
the grooves of the profiles) and wingnuts (Fig. 11).
Fig. 11: Acrylic sheet connected to extrusion profile using hammer-head screw and wingnut.
Fix second stepper motor to the stepper motor mount (Section 3) using M3 screws, and the
mount to a 10 cm extrusion profile using M5 screws, washers and hammer nuts (Fig. 12).
Fig. 12: Stepper motor fixed to extrusion profile using stepper motor mount (Section 3).
Connect carousel to stepper motor shaft (Fig. 13).
Fig. 13: Carousel connected to stepper motor by shaft.
Finally, the carousel needs to be fixed to the gantry. This needs to be done so that the hole in the
acrylic base is on the reach of each hole in the carousel. Also, the carousel must be close, but not
touching, the acrylic. Place the stepper motor attached to the carousel on the acrylic sheet. At this
point, they should already be connected to a 10 cm extrusion profile bar (Fig. 12). Place 3 sheets
of normal printing paper between the carousel and the sheet, and then fix the 10 cm profile to the
25 cm profile (gantry between the two acrylic supports) using a 2028 bracket (Fig. 12). Remove
the paper sheets. The table should be fully assembled by then.
5.2: Assembling the purging pipe
Remove the levers that come with the valves, and connect custom-made levers to the ball valves
using the nuts that come with the valves (Fig. 14). Ensure that the levers make 90o with the main
valve body when the valve is closed, and are parallel to the main valve axis when it is opened.
Fig. 14: Valve with lever replaced by a custom-made one. This lever is made from a steel
striping (middle of the figure), being cut and drilled according to the necessary dimensions.
Here, the existing holes on the stripping were used as a starting point, and matching holes with
enough diameter (here, 3 mm) were also drilled at the servo arm (right of the figure). The
rotation center of the lever and the arm must also match when assembled (Fig. 15).
Connect the servo arm to the lever using M3 screws, nuts and custom-made spacers listed in
Section 3 (Fig. 15). Make sure that the M3 screws are connected at opposing orientations (Fig.
15). This ensures that the levers remain connected even after many repeated movements.
Fig. 15: Valve lever connected to servo arm. Notice screws connected at opposing orientations.
Before connecting the servo motor to its arm, make sure the shaft is at a known position that
matches that of the arm-lever on the valve. For example, if the lever is making 90o with the
valve, connect the servo at position 0o. The servo position can be setup using the control board,
as described in section 6.2.
A second connection point between servo and valve is needed to make it possible for the servo to
actuate the valve. Using M4 screws, nuts, washers, spacers (Section 3), and the servo-valve
connector (Section 3), connect the servo to the valve (Fig. 16).
Fig. 16: Servo motor fully connected to ball valve.
Connect the valves to the remaining parts of the purging pipe (Fig 17). Except for the topmost
connection above the topmost valve, connections must be leak free. This can be ensured using
either O-rings or Teflon tape. Gas leaks can be detected using appropriate devices. For helium,
for example, a helium sniffer is very useful. For the topmost connection, use a connector which
has no potential surfaces for samples to land on top and not fall to the surface of the valve. This
is particularly important when analyzing small samples, which can potentially get trapped on
such surfaces.
Fig. 17. Purging pipe fully assembled.
The connector between the lower and middle valve (a T piece) has a large dead space at the
perpendicular connection. It is useful to add a sieve (Section 3) to avoid samples to settle at this
part of the connector (Fig. 18).
Fig. 18: Sieve to be placed inside the T piece to avoid samples to settle at the perpendicular
connection.
Once fully assembled, connect the purging pipe to the elemental analyzer (Fig. 17).
5.3: Attaching the control boards and connecting the motors to the board
The control board is attached to one of the legs of the sampling table. The exact position must be
decided when both the table and the purging pipe are placed on the elemental analyzer (Fig. 1),
so that the wiring of the servo motors can reach the board without tension (Fig. 19).
Fig. 19: Control board fixed to the autosampler, positioned on reach of servo wires.
Connect stepper motors of each axis (X for gantry, and Y for carousel) to the appropriate stepper
motor drive (Fig. 20). Importantly, connect the stepper motor drivers at the correct orientation
(Fig. 20).
Fig. 20: Stepper motor wires connected to the control board.
Connect servo motors to the appropriate slots (Fig. 21).
Fig. 21. Servos connected to positions 0, 2 and 3. These positions are arbitrary and match the
code in Supplementary Information 2. Different positions can be chosen, as long as the code is
modified accordingly.
After motors are all wired to the board, the board can be powered with the power supply and
connected to the computer using the USB cable (lower right corner of Fig. 21).
6. Operation Instructions
6.1 Motor control
The firmware (OsmarMarlin, available from https://osf.io/qt9pg/ or
https://doi.org/10.17605/OSF.IO/UTZSE) must be uploaded to the board using the Arudino IDE
(available from https://www.arduino.cc/en/main/software). For this, the board must be connected
to a computer via USB, and a BAUD rate of 115200 should be used when uploading the
firmware. This version of Marlin is an updated version of the one presented for previous
autosamplers [29,30], in order to become compatible with the current (at the time of writing)
version of Arduino. On top of the modifications listed in these previous papers, two others were
added to the configuration tab of this version to enable servo motor control: 1) the line “#define
NUM_SERVOS” became #define NUM_SERVOS 4 ; 2) the line “#define SERVO_DELAY
{300}” became #define SERVO_DELAY {300, 300, 300, 300}.
Motor control is done using G-code via the program Hype!terminal (available from
https://osf.io/p7gh5/ or https://doi.org/10.17605/OSF.IO/UTZSE). The serial connection settings
are: 115200 for Baud rate, 8 data bits, none for parity, 1 stop bit, and none for flow control. The
G-code dialect used is that for Marlin, thus including M commands [29]. The stepper motors are
controlled using the G1 command: G1<axis><distance><speed><movement speed>. For
example, the command G1X10F500 means that the motor controlling the X axis will move 10
steps at 500 steps per minute. Importantly: before starting sending G commands, it is
recommended that the command M121 is sent (just once after connection between board and
computer is stablished), so that the full range of step values (including negative ones) can be
reached.
Servo motors are actuated using the command M280, which has the usage M280P<motor
number>S<angle in degrees>. The motor number can be 0 to 3, and the angle 0 to 180. For
example, the command M280P0S130 will turn the shaft of the motor 0 (motor number
corresponds to valve number, as explained before) to the angle 130o in relation to its origin.
For the carousel presented in Table 1 and Fig. 4, and for the stepper motors used here, we
used feed rates of 50 (X axis) and 25 (Y axis). The X movement between carousel circles was
found to be 1.1225. Therefore, to move from the initial circle (the most external one) to the next,
the command was G1X1.1225F50. For the next, the step value was added of 1.1225, thus being
G1X2.245F50. For the Y axis, the space between holes changes for each circle. For the most
external the step was 0.09333, for the innermost 0.44167. Thus, for the most external circle the
command from the first to the second hole was G1Y0.09333F25. If these values do not work for
your setup, determine one by one by trial and error. For both X and Y axes, the best approach is
to do a full movement (for example, from 0 to the maximum linear X range, or from 0 to the
maximum Y circular range for each circle) and divide by the number of holes in each case.
It is important to ensure that movement is consistent and reproducible. We have observed
that if the control board is shut off and on again, the first movement of a stepper motor jitters.
Thus, an adjustment is needed. This can be done using the command G92. For example, to
determine that a given position is 0 for the Y axis, send G92Y0.
6.3 Integration with elemental analyzer
The autsosampler is synchronized to the elemental analyzer using AutoIt [24]. A script is
written which coordinates commands between the software controlling the autosampler
(Hype!terminal) and the software controlling the elemental analyzer (in this case, Isodat by
Thermo Fisher). For a group of samples, the first sample to be measured is placed inside the
purging pipe, directly on top of the top valve, which should be closed, as the other valves (Fig.
3). When isodat starts a measurement for the elemental analyzer, the status bar shows a message
which AutoIt uses as a trigger for the autosampler. Once this trigger is received, the following
actions take place: 1) the top valve opens, 2) the carousel spins (unless it is the first sample in a
sequence, which will be already inside the purging pipe, as explained before), 3) the top valve
closes; 4) the top valve and middle valve partially open, and remain opened for 60 s; 5) the top
valve closes, and the middle valve opens, so that the sample falls on top of the bottom valve; 6)
the middle valve closes; 7) the bottom valve opens, delivering the sample to the elemental
analyzer; 8) the bottom valve closes, and the cycle can be repeated. The full code for all the steps
is provided in Supplementary information 2.
The servo motors employed to actuate the purging pipe were capable of relatively high
torque (1.94 N m). This torque demanded a relatively high current load which, if maintained for
more than a few seconds (typically 60 s), caused problems of communication between the
control board and the computer. This was dealt with by closing and opening communication
between board and computer after at most three commands sent to the servos. This procedure cut
the power supply and stopped servos from holding torque unnecessarily. The repeated execution
of this procedure caused the COM port communication software (Hype!terminal) to crash, which
was avoided by terminating and re-starting the program once every sample run. Despite
considerable complexity being added to the operating of the autosampler, these steps ensured
flawless sampling. A different combination of control board, communication software, ball valve
and / or servo motor may prove more adequate and less demanding of computing and power
resources, thus allowing a simpler operation of the system. Details of the procedure described
here are given with the rest of the programming code in Supplementary Material 2.
6.4 Routine operation and maintenance
The most common procedure that demands partial disassembly of the autosampler is the
removal of ashes from the elemental analyzer reactor, which must be done after a sufficient
number of samples has been measured. It is not necessary to disconnect the servo motors from
the board for this procedure, but, if it is decided to do so, it is advisable to firstly cut the power
supply to the board. The purging pipe is then left at a safe position, and pushed along with the
sampling table away from the top of the reactor. The reactor can now be pulled out of the
furnace. The reactor can be hot, so care is needed during this operation. After ash removal, the
reactor can be reinserted in the furnace, and the sampling pipe re-connected to it. Leaks need to
be re-checked and, if none are found, the elemental analyzer can be re-started. If the power
supply to the autosampler control board was cut, it must be re-connected, and the positions for
the stepper motors verified, in particular the movement for the Y axis, which may jitter at the
first execution.
It is advisable that sample remains are periodically cleaned from the autosampler. The
carousel and its supporting table can be gently brushed after a sample run to remove small specs
of samples, and occasionally rinsed with acetone if needed. Moving the carousel on its X and Y
axes can help with the cleaning procedure. If this is done manually, it is necessary to re-set the
positions of the axes on the controlling software to ensure proper operation.
It is good practice to always verify motor operation before starting a run to avoid simple
accidents. This verification must always be done without any sample in the carousel.
If large samples are introduced in the autosampler, it is possible that they will get trapped
by one of the ball valves and be destroyed. If this happens, it is possible that the valve becomes
stiff and the servo cannot turn it properly. Disassembling the purging pipe and cleaning the valve
is necessary in these cases. If the valve is still stiff after cleaning, it needs to be replaced, because
continued operation of a stiff valve can ultimately damage the servo moving it. Therefore, it is
good idea to have spares of these valves (and, in fact, of all components) for when such problems
happen. If this kind of problem happens several times with the same valve, eventually the servo
motor will also become damaged, and need to be replaced. Therefore, spare servos should be
kept as well.
It is advisable to routinely (at least once every two weeks) inspect all screw connections in
the purging pipe, and fasten the loose ones. Loose connections will result in incomplete valve
movement. If the bottom valve does not close completely, a gradual increase in the N2
background from sample to sample, accompanied by a delay and broadening of sample peaks,
are usually observed.
At the moment that the lower valve opens for the sample so that it falls into the reactor, the
flow of the carrier gas is affected. If there is any blockage in the line, backpressure can push the
contents of the reactor all the way to the top of the reactor, at its junction with the valve. This
problem is avoided by keeping the gas lines unobstructed.
7. Validation and Characterization
The autosampler was evaluated as an accessory for a Thermo Fisher Flash EA elemental
analyzer coupled to a Thermo Fisher isotope ratio mass spectrometer. Samples were measured
for N and C content using a modified Dumas combustion procedure [1,2]. Samples were
combusted under an oxygen pulse at 1020oC in an oxidation reactor, where helium flowing at
150 mL min-1 carried the gases from combustion through chromium oxide pellets, and then
through silvered cobalt ones. The gases were then transported to a reduction reactor kept at
650oC and containing reduced copper wire pellets. The gas mixture was carried through a water
trap (magnesium perchlorate), and then through a chromatographic column where gases were
separated, notably N2 from CO2, because they are the gases being quantified for N and C content,
respectively. Then the gases were carried through an interface (Conflo IV, by Thermo Fisher),
where the gases were diluted so that they reached the mass spectrometer at 0.3 mL min-1. At the
mass spectrometer, different protonated N2 (or CO2) isotopic configurations were deviated using
a magnetic field and collected in faraday cups, where their relative abundances were quantified.
The main potential problem of the sampling procedure is air intrusion into the reactor
together with the sample, which would interfere with measurements of gases similar to those
abundant in the atmosphere, like N2 and O2. Thus, air input due to valve movement was
evaluated by comparing it to operation of the elemental analyzer without any movement of the
autosampler (Fig. 22). When used like that, the elemental analyzer modifies the flow by allowing
an oxygen pulse into the helium stream, which slightly disturbs the baseline and adds a very
small amount of N2, which is visible as a small peak latter in the chromatogram. When the
autosampler was used, the perturbation of the baseline and the N2 peak were both slightly larger
than when only the elemental analyzer was used (Fig. 22). However, such perturbations were
very small in both cases, which is clear when the peaks from real samples are compared to the
autosampler working with no sample (Fig. 23). There was also no effect on detector sensitivity,
as samples with as little as 9 µg of N generated peaks with enough amplitude for useful isotopic
measurements.
Fig. 22: Comparison between chromatograms generated by elemental analyzer when only
the elemental analyzer is used, and when the elemental analyzer is used together with the
autosampler. Both chromatograms are shown with very large augmentation, and perturbations on
the base line are very small in both cases.
Fig. 23: Comparison of chromatograms when the autosampler delivers no sample, and
when samples with small and larger amounts of material are delivered. The first and last two
peaks (the four rectangles) are reference gas peaks, the first two for N2, and the last two for CO2.
The gaussian peaks are for the analyzed samples, the first one for N2, and the second one for
CO2.
This test demonstrated that the autosampler fulfills the need for avoiding air contamination
in the measurements. These results were obtained using the maximum flow for the purging gas
(300 mL min-1) that the elemental analyzer permits. Even at this high flow rate helium
consumption was low, because the valves are kept closed during most of the operation time.
Despite the negligible air influence on measurements, the valve movement caused a
perturbation on the baseline (Fig. 22). This perturbation did not affect the sample peaks
themselves, which came several seconds afterwards (Fig. 22). For isotopic measurements, which
demand the measurement of reference gas peaks (the rectangular peaks in Fig. 23), valve
opening was timed to happen only after these peaks for N2 had been finished in the
chromatogram, thus ensuring no influence of valve movement on their measurement.
By the time this text was written, more than 6,000 samples have been introduced in the
elemental analyzer by the autosampler, generating useful scientific data. This has spanned more
than a year of almost daily use of the autosampler. Routinely we have observed precision of 1%
for both N and C contents, and 0.15 ‰ and 0.1 ‰ for N and C stable isotopes. These values are
in accordance with the instrument specification, and demonstrate that the autosampler has not
added significant errors to the measurements. Importantly, the autosampler was demonstrated
solely for C and N analyzes of solids, but it could also be useful for other elements, like sulfur,
oxygen and hydrogen, which are done using similar analytical equipment [31-34,1,35].
The carousel employed here was designed with the specific needs of our laboratory in
mind: it holds 95 samples for a single run, which allows almost 24 hours of unassisted operation.
Also, sample holes are wide enough to accommodate relatively large samples, which in our
laboratory consist of sandy soils or sediments, and glass fiber filters of natural waters. More
samples could be arranged for a single run, if so desired, by simply enlarging the carousel
diameter, or making narrower sample holes. The provided 3D-printing file (Section 3) can be
freely modified for such purposes.
The purging pipe was built using mostly of-the-shelf parts, but most of them could be
replaced by custom-made, perhaps 3D-printed parts, so that the whole structure could be made
shorter and simpler. These custom-made parts would need to be either made of metal, or of a
heat-resistant, non-porous plastic in order to function properly.
Compared to commercial models (Costech Zero Blank Autosampler and Thermo Fisher
A200s Autosampler), the model presented here has several advantages (Table 1). It is clear from
there that the autosampler presented here is a low-cost and viable alternative to existing
commercial models.
Table 1: Comparison between the autosampler presented here and two commercial models.
Parameter
This autosampler
Costech Zero Blank
Autosampler
Thermo-Fisher
MAS200r
Number of
samples per tray
95; can be expanded
without modifying
sample size if a larger
tray is designed; or a
larger tray can be built,
along a larger table, so
that a larger number of
large samples can be
49 for 6 mm samples
32; more samples can
be added if extra trays
are pilled upon each
other, but these need
to be purchased
separately
accommodated
Maximum sample
size
Sphere with 10 mm in
diameter
Sphere with 6 mm in
diameter when using a
tray for 49 samples; it
can take larger samples
but then the tray needs
to be swapped with one
for less samples at once
Sphere with 10 mm in
diameter, but only if
purging flow is largely
increased
Sample adding
procedure
Samples need to be
placed inside their
respective holes in the
carousel
Samples need to be
placed inside carousel,
and then a flushing
procedure lasting about
10 minutes is needed. If
more samples need to
be added, the flushing
needs to be repeated
again.
Samples need to be
placed inside their
respective holes in
carousel and a lid
needs to be placed
over the tray. If the lid
is forgotten, large air
blanks are introduced
in the measurement.
Cost
AU$540 plus extras
AU$20,000
AU$10,000
Replacement parts
3D-printed or readily
available from
hardware stores
Proprietary
Proprietary
Compatibility
Universal via AutoIt
Proprietary, only for
designed partner
companies
Proprietary, only for
designed partner
companies
8. Acknowledgements
We are grateful to Prof. Bradley Eyre, Southern Cross University, for allowing access to
analytical equipment. This research did not receive any specific grant from funding
agencies in the public, commercial, or not-for-profit sectors.
9. Declaration of interest
There are no conflicts of interest.
10. Human and animal rights
The work did not involve human or animal subjects.
References:
[1] P.A. de Groot, Handbook of stable isotope analytical techniques, vol. 2. Elsevier, (2009)
[2] B. Fry, W. Brand, F.J. Mersch, K. Tholke, R. Garritt, Automated analysis system for coupled d13C and
d15N measurement. Analytical chemistry, 64, 288-291 (1992).
[3] M.A. Teece, M.L. Fogel, Preparation of ecological and biochemical samples for isotope analysis. In:
Groot, P.A. (ed.) Handbook of stable isotope analytical techniques, vol. 1. pp. 177-202. Elsevier,
Amsterdan (2004)
[4] T. Preston, N.J.P. Owens, Interfacing an automatic elemental analyzer with an isotope ratio mass-
spectrometer - the potential for fully automated total nitrogen and N-15 analysis. Analyst, 108,
971-977 (1983). doi:10.1039/AN9830800971
[5] R. Michener, K. Lajtha, Stable isotopes in ecology and environmental science. Wiley, (2007)
[6] J. Hoefs, Stable isotope geochemistry. Springer-Verlag, Berlin (2009)
[7] B. Fry, Statble Isotope Ecology. Springer, New York (2006)
[8] W. Meier-Augenstein, Stable isotope forensics: methods and forensic applications of stable isotope
analysis. Wiley, (2018)
[9] L.I. Wassenaar, K.A. Hobson, L. Sisti, An online temperature-controlled vacuum-equilibration
preparation system for the measurement of δ2H values of non-exchangeable-H and of δ18O
values in organic materials by isotope-ratio mass spectrometry. Rapid Communications in Mass
Spectrometry, 29, 397-407 (2015). doi:10.1002/rcm.7118
[10] M. Wooller, B. Collins, M.L. Fogel, The elemental analyzer sample carousel: loading an autosampler
made easy. Rapid Communications in Mass Spectrometry, 15, 1957-1959 (2001).
doi:10.1002/rcm.468
[11] NOAA, Sub-surface automated sampler. https://www.coral.noaa.gov/accrete/sas/, (2019).
[12] M.Y. Kamogawa, M.A. Teixeira, A low cost autosampler for flow injection analysis. Quimica Nova,
32, 1644-1646 (2009). doi:10.1590/S0100-40422009000600048
[13] L. Vault, E. Scarano, G. Tosello, A. Boisen, Fully replicable and automated retention measurement
setup for characterization of bio-adhesion. HardwareX, 6, e00071 (2019).
doi:10.1016/j.ohx.2019.e00071
[14] A.H. Grange, An inexpensive autosampler to maximize throughput for an ion source that samples
surfaces in open air. Environmental Forensics, 9, 127-136 (2008).
doi:10.1080/15275920802115860
[15] S. Steffens, L. Nüßer, T.-B. Seiler, N. Ruchter, M. Schumann, R. Döring, C. Cofalla, A. Ostfeld, E.
Salomons, H. Schüttrumpf, H. Hollert, M. Brinkmann, A versatile and low-cost open source
pipetting robot for automation of toxicological and ecotoxicological bioassays. Plos One, 12,
e0179636 (2017). doi:10.1371/journal.pone.0179636
[16] S.A. Longwell, P.M. Fordyce, micrIO: An Open-Source Autosampler and Fraction Collector for
Automated Microfluidic Input-Output. BioRxiv, in press (2019). doi:10.1101/655324
[17] S. Eggert, D.W. Hutmacher, In vitro disease models 4.0 via automation and high-throughput
processing. Biofabrication, 11, 043002 (2019). doi:10.1088/1758-5090/ab296f
[18] S.-H. Chiu, P.L. Urban, Robotics-assisted mass spectrometry assay platform enabled by open-source
electronics. Biosensors and bioelectronics, 64, 260-268 (2015). doi:10.1016/j.bios.2014.08.087
[19] C.-L. Chen, T.-R. Chen, S.-H. Chiu, P.L. Urban, Dual robotic arm "production line" mass spectrometry
assay guided by multiple Arduino-type microcontrollers. Sensors and Actuators B: Chemical,
239, 608-616 (2017). doi:10.1016/j.snb.2016.08.031
[20] A. Booeshaghi, E.V. Beltrame, D. Bannon, J. Gehring, L. Patcher, Principles of open source
bioinstrumentation applied to the poseidon syringe pump system. Scientific Reports, 9, 12385
(2019). doi:10.1038/s41598-019-48815-9
[21] C.D. Hawker, M.R. Schlank, Development of standards for laboratory automation. Clinical chemistry,
46, 746-750 (2000).
[22] V. Cerdá, G. Ramis, An introduction to laboratory automation. John Wiley & Sons, New York (1990)
[23] H. Bär, R. Hochstrasser, B. Papenfuß, SiLA: basic standards for rapid integration in laboratory
automation. Journal of laboratory automation, 17, 86-95 (2012).
[24] M.C. Carvalho, Practical laboratory automation made easy with AutoIt. Wiley VCH, (2016)
[25] K. Juul-Madsen, H. Zhao, T. Vorup-Jensen, P. Schuck, Efficient data acquisition with three-channel
centerpieces in sedimentation velocity. Analytical biochemistry, in press (2019).
doi:10.1016/j.ab.2019.113414
[26] M.L. Scherholz, J. Forder, I.P. Androulakis, A framework for 2-stage global sensitivity analysis of
GastroPlus™ compartmental models. Journal of pharmacokinetics and pharmacodynamics, 45,
309-327 (2018). doi: 10.1007/s10928-018-9573-1
[27] M.C. Carvalho, Integration of analytical instruments with computer sripting. Journal of laboratory
automation, 18, 328-333 (2013).
[28] M.C. Carvalho, B.D. Eyre, A low cost, easy to build, portable, and universal autosampler for liquids.
Methods in oceanography, 8, 23-32 (2013). doi:http://dx.doi.org/10.1016/j.mio.2014.06.001
[29] M.C. Carvalho, R.H. Murray, Osmar, the open source microsyringe autosampler. HardwareX, 3, 10-
38 (2018). doi:https://doi.org/10.1016/j.ohx.2018.01.001
[30] M.C. Carvalho, C.J. Sanders, C. Holloway, Auto-HPGe, an autosampler for gamma-ray spectroscopy
using high-purity germanium (HPGe) detectors and heavy shields. HardwareX, 4, e00040 (2018).
doi:doi.org/10.1016/j.ohx.2018.e00040
[31] A. Giesemann, Jager H.-J., Norman A. L., K.H. R., B.W. A., On-line sulfur-isotope determination using
an elemental analyzer coupled to a mass spectrometer. Analytical chemistry, 66, 2816-2819
(1994). doi:10.1021/ac00090a005
[32] B.E. Kornexl, M. Gehre, R. Hofling, R.A. Werener, On-line 18O measurements of organic and
inorganic substances. Rapid Communications in Mass Spectrometry, 13, 1685-1693 (1999).
doi:10.1002/(SICI)1097-0231(19990830)13:16<1685::AID-RCM699>3.0.CO;2-9
[33] S.D. Kelly, I.G. Parker, M. Sharman, M.J. Dennis, On-line quantitative determination of 2H/1H isotope
ratios in organic and water samples using an elemental analyser coupled to an isotope ratio
mass spectrometer. Journal of Mass Spectrometry, 33, 735-738 (1998). doi:10.1002/(SICI)1096-
9888(199808)33:8<735::AID-JMS692>3.0.CO;2-I
[34] R.A. Werner, B.E. Kornexl, A. Rossman, H.-L. Schmidt, On-line determination of 18O values of
organic substances. Analytica Chimica Acta, 319, 159-164 (1996). doi:10.1016/0003-
2670(95)00468-8
[35] Z.D. Sharp, V. Aturodei, T. Durakiewicz, A rapid method for determination of hydrogen and oxygen
isotope ratios from water and hydrous minerals. Chemical Geology, 178, 197-210 (2001).
doi:10.1016/S0009-2541(01)00262-5
