DIONEX ICS-3000 Ion Chromatography (IC) system
Standard Operating Procedures (SOPs)
I. ICS-3000 System Overview
1. System Overview
Our new Dionex ICS-3000 Ion Chromatography (IC) system has 4 main components: Auto
sampler (AS); Detector/ Chromatography (DC); Dual Pump (DP); Eluent Generator (EG).
Chromeleon Tablet Station (computer and software) was installed to control t. Please be familiar
with these components before starting, check Figure 1 for details.
Figure 1 Dionex ICS-3000 Ion Chromatography (IC) system Components
II. Startup Procedures
1. IC System Startup
*only applicable if system power is off for maintenance or for long term vacancy.
Before you can operate the system through Chromeleon software, you should make sure all IC
components have been properly started and are in good working status. The steps to startup IC
system are shown as follows:
1) Make sure enough eluent is in the eluent reservoir (for sugar analysis, mili-Q water is used
as eluent)
2) Press the power switches (1-2 seconds) to turn on each component in sequence of: Auto
sampler (AS)-> Dual Pump (DP)->Detector/ Chromatography (DC)->Eluent Generator (EG);
3) Wait for 1 minute for heat-up;
4) Make sure all components are in good working status (If there is red alarm light on or
flashing, please contact Responsible User or Barbara for assistance).
5) Turn on Tablet Station (a tablet laptop mounted on an articulating arm)
6) Click ‘start’ -> ‘server monitor’ ->’start’ button to start Chromeleon server (Figure 2), it
will take 1-2 minutes for server to be ready;
Figure 2 Starting Chromeleon Server
7) Please check the status bar on the front of each component, make sure Detector/
Chromatography (DC); Dual Pump (DP) and Eluent Generator (EG) are connected (green
light, Figure 3);
Figure 3 Status bar on the front of each component
8) Click ‘start’ -> ‘Chromeleon’ to start Chromeleon software;
9) Click ‘view’ then‘Default Panel Tabset’ button
on toolbar then select ‘my computer’ –
>’ Chromeleon Server’ ->’OK’ to initialize the Panel Tabset (Figure 4),
10) Click on the ‘expand icon’ on ‘ncsu_1’ (left side) to enlarge the window;
Figure 4 Open Default Panel Tabset
11) The panel tabset opens to the ‘Home’ panel by default. This panel displays basic status
information for each instrument in the system (Figure 5).
Figure 5 ‘Home’ panel
2. Degas Eluent (Optional)
Although the new Dionex has integrated a degas unit in its pump, it is still recommended to
degas your eluent (mili-Q water for sugar analysis) before loading to the system. Please estimate
the volume of eluent you will need for your sequence run. For sugar analysis, the pump flow
rate is set to 1.0 ml/min. One full eluent reservoir can approximately support 30 hours running
time. To calculate the amount of mili-Q water needed:
Samples: 30 min × # of samples
Cleaning: 30 min × # of cleanings
Shutdown: 30 min × 1
Total Minimum Volume: =
=
=
=
To degas eluent (operating in old Dionex system):
1) Attach degassing line to bottle eluent (line stored in front of ED40);
2) Open compressed helium tank and set at 11 psi;
3) Pull out black knob on front of the ED40 electrochemical detector and set to 3-4 psi on
black scale, snap back in;
4) Allow to purge for 30 min;
5) Close down everything in reverse order, remove degassing line.
3. Prime Pump
The pump must be primed each time after refilling eluent reservoir OR if the components are
off OR the system is idle for more than 2 days. It is suggested you prime the pump every time
you start a new sequence run.
1) On panel tabset switch to ‘Gradient Pump’ panel (Figure 6);
Figure 6. Gradient Pump Panel
2) Click ‘Prime’ button, you will be instructed to open ‘Prime Valve’ before continuing
(Figure 7);
Figure 7
3) Open ‘Prime Valve’ on PUMP 1 by turning it one-half turn counter-clockwise (Figure 8);
Figure 8. Prime valve
4) Click ‘OK’ to start priming, by default it will take 5 min @ a flow rate of 6 ml/min;
5) Prime twice if air bubbles still exist in inlet tubing.
6) Close ‘Prime Valve’ by finger tightening.
4. Baseline Stabilization to stabilize
Before running samples, please allow baseline to stabilize.
1) Switch back to ‘Home’ panel (Figure 9);
Figure 9. Home panel
2) Click ‘Start up’ button, wait for 30 seconds or until pump pressure is stablized;
3) Click ‘Start up’ again to ensure the status of EG component is ‘On’ (the eluent generate
will not start if pump pressure is below 1000 psi)
4) Make sure all components are working properly;
5) Please check eluent concentration on eluent generator ‘EluGen-OH’ display, for
Aplication I (non-woody material sugar analysis, by default), 18mM should be used.
Please check required eloquent concentration in table 1. Click ‘Eluent Generator’ panel,
and manually set to your desired concentration;
Table 1. Eluent concentration for different applications
Applications
I. Non-woody materials
II. Wood (soft and hard wood)
III. Cellobiose
OH concentration
18 mM
2.5 mM
50 mM
Program
sugar_nonwood.pgm
sugar_wood.pgm
Cellobiose.pgm
6) Click ‘Control’ menu -> ‘Acquisition on’-> ‘OK’;
7) Switch to ‘Status’ panel, allow to run for 30 min to ensure flat baseline;
8) Click ‘Control’ menu -> ‘Acquisition off’ to stop acquisition before starting your sample
run.
Application I: Quantification of Non-wood Structural Carbohydrates
Precaution: Application I can be only applied to non-wood materials, which has no or trace amount of
mannan (polymer of the sugar mannose). Non-wood materials usually refer to raw or pretreated nonwood lignocellulosics such as grasses, stalks, stover or starch. The limitation of this procedure is its
inability to separate mannose from xylose. However, it provides a fast (~30min per sample) and stable
separation and quantification of other major structural sugars i.e. arabinose, rahmnose, galactose,
glucose, and xylose.
I. Sample Preparation
1. General Guideline for Preparing Sugar Samples:
1) Neutralize sample if necessary (only for acid hydrolyzed samples, large sample
size >10ml may be needed to perform neutralization); Use barium hydroxide, Ba(OH)2 to
neutralize acidic sample to a pH range 5-6;
2) Dilute sugar samples to ~ 400 ug/ml glucose (or xylose, whichever is the most abundant
sugar in sample) with mini-Q water (please refer to example calculation);
3) Supplement with internal standard (fucose) by mixing 2ml of 200 ug/ml fucose with 2ml
of diluted sugar samples; the final concentration of 100ug.
4) Filter sugar mixture through 0.2 um nylon filter into 1ml autosampler vial and then cap
with a pre-slit septum;
5) Store prepared samples @ 4C in refrigerator before loading into auto-sampler (the sugar
samples may be stable @ 4C for less than 1 week, for long term storage, the samples
should be stored in a freezer @-20C and removed when needed. Thaw and vortex
frozen samples prior to use )
Example Calculation:
a.
b.
Acid hydrolyzed sample: The NREL two step acid hydrolysis procedure was followed to perform lignin and
structural carbohydrate assay, which means 0.3 OD gram solid samples were subjected to 3 ml 72% H2SO4
and then diluted with 84 ml DI water, making the total volume of 87 ml. Assuming 50% cellulose in solid
sample, the total estimated glucose concentration in liquid fraction will be 0.3×50%×1.1×106/87 ~= 2000
ug/ml. To make the desired concentration ~ 200 ug/ml, a 10× dilution is recommended.
Enzymatic hydrolyzed sample: 5 OD gram sample was subjected to enzymatic hydrolysis in a total volume of
100 ml (5% solid loading). Assuming 70% cellulose in solid sample and a 85% cellulose conversion, the total
estimated glucose concentration in liquid fraction will be 5×70%×85%×1.1×106/100 ~= 32,000 ug/ml. To
make the desired concentration ~ 200 ug/ml, an 160× dilution is recommended.
2. Sugar Calibration Standard Preparation:
1) Prepare sugar stock solution:
a) weigh out ~0.5g of each sugar (fucose, arabinose, rahmnose, galactose, glucose, and
xylose) and dry at 45C overnight using vacuum oven;
b) accurately weigh 0.1±0.001 g of each OD sugar sample and transfer into 100 ml
volumetric flasks separately (record the weights);
c) add mili-Q water up to 100 ml to make 10 mg/ml sugar stock solution for each sugar;
d) keep @ 4C for short term or freeze @-20C for long term storage.
2) Prepare 4 levels of calibration standards by following the dilutions in Table 1, dilute with
50 ml volumetric flasks;
100mg/ml
stock solution
Fucose
Arabinose
Rahmnose
Glactose
Glucose
Xylose
Mini-Q water
Table 1. Recipe for 4 level calibration standards
Level #1 Level #2 Level #3 Level #4 Note: final concentration
level 1 – 4 (ug/ml)
1000 ul
125 ul
50 ul
125 ul
250 ul
250 ul
to 50 ml
1000 ul
250 ul
100 ul
250 ul
500 ul
500 ul
to 50 ml
1000 ul
375 ul
150 ul
375 ul
750 ul
750 ul
to 50 ml
1000 ul
500 ul
200 ul
500 ul
1000 ul
1000 ul
to 50 ml
200 ug/ml for all levels
25, 50, 75, 100 ug/ml
10, 20, 30, 40 ug/ml
25, 50, 75, 100 ug/ml
50, 100, 150, 200 ug/ml
50, 100, 150, 200 ug/ml
In 50 ml volumetric flasks
3) Filter calibration standards through 0.2 um nylon filter into 1ml autosampler vial and
then cap with a pre-slit septum;
4) Store prepared samples @ 4C in refrigerator before loading into auto-sampler (the sugar
samples may be stable @ 4C for less than 1 week, for long term storage the samples
should be stored in a freezer @-20C and remove when needed. Thaw and vortex frozen
samples prior to use )
3. Sugar Recovery Standard (SRS) Preparation:
1) Use the sugar stock solution to prepare 4 levels of sugar recovery standards (SRS) by
following the dilutions in Table 2, dilute in 50 ml volumetric flasks;
100mg/ml
stock solution
Fucose
Arabinose
Rahmnose
Glactose
Glucose
Xylose
Table 2. Recipe for 4 levels sugar recovery standards
Level #1 Level #2 Level #3 Level #4 Note: final concentration
level 1 – 4 (ug/ml)
1000 ul
125 ul
50 ul
125 ul
250 ul
250 ul
1000 ul
250 ul
100 ul
250 ul
500 ul
500 ul
1000 ul
375 ul
150 ul
375 ul
750 ul
750 ul
1000 ul
500 ul
200 ul
500 ul
1000 ul
1000 ul
200 ug/ml for all levels
25, 50, 75, 100 ug/ml
10, 20, 30, 40 ug/ml
25, 50, 75, 100 ug/ml
50, 100, 150, 200 ug/ml
50, 100, 150, 200 ug/ml
72% H2SO4
Mini-Q water
1724 ul
to 50 ml
1724 ul
to 50 ml
1724 ul
to 50 ml
1724 ul
to 50 ml
Final concentration 4% H2SO4
In 50 ml volumetric flasks
2) Transfer prepared standards into serum bottles, cap with rubber stoppers and
aluminum easy-peel caps;
3) Autoclave @ 121C (~15psi) for 1h;
4) After completely cooling down, use barium hydroxide, Ba(OH)2 to neutralize acidic
sample to a pH range 5-6;
5) Filter neutralized SRS standards through 0.2 um nylon filter into 1ml auto-sampler vial
and then cap with a pre-slit septum;
6) Store prepared samples @ 4C in refrigerator before loading into auto-sampler (the sugar
samples may be stable @ 4C for less than 1 week, for long term storage the samples
should be stored in a freezer @-20C and removed when needed. Thaw and vortex
frozen samples prior to use )
II. Calibration and Verification
Pending…
III. Sample Run
1. Please follow Startup Procedures to prepare Dionex system to be ready for
sample running.
2. Switch to ‘project browser’ by clicking ‘Windows’ -> ‘Toshiba_user…’;
3. On the left, find your desired directory (you may create your own directory to manage
all your data);
4. Copy ‘non_wood’ from ‘application_backup’ directory to your own directory;
5. Select ‘non_wood’ from your own directory, you may rename it;
6. Edit the sequence and save it;
7. Click ‘Batch’ -> ‘Start’, on the popup window, click ‘verify’, make sure there are no alarm
messages, then click ‘OK’ to start sample run.
IV. Calculations
Pending…