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 OH concentration ProgramI. Non-woody materials 18 mM sugar_nonwood.pgmII. Wood (soft and hard wood) 2.5 mM sugar_wood.pgmIII. Cellobiose 50 mM 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 non-wood 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. 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.

b. 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;

Table 1. Recipe for 4 level calibration standards100mg/ml stock solution

Level #1 Level #2 Level #3 Level #4 Note: final concentrationlevel 1 – 4 (ug/ml)

Fucose 1000 ul 1000 ul 1000 ul 1000 ul 200 ug/ml for all levels

Arabinose 125 ul 250 ul 375 ul 500 ul 25, 50, 75, 100 ug/ml

Rahmnose 50 ul 100 ul 150 ul 200 ul 10, 20, 30, 40 ug/ml

Glactose 125 ul 250 ul 375 ul 500 ul 25, 50, 75, 100 ug/ml

Glucose 250 ul 500 ul 750 ul 1000 ul 50, 100, 150, 200 ug/ml

Xylose 250 ul 500 ul 750 ul 1000 ul 50, 100, 150, 200 ug/ml

Mini-Q water to 50 ml to 50 ml to 50 ml to 50 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;

Table 2. Recipe for 4 levels sugar recovery standards

100mg/ml stock solution

Level #1 Level #2 Level #3 Level #4 Note: final concentrationlevel 1 – 4 (ug/ml)

Fucose 1000 ul 1000 ul 1000 ul 1000 ul 200 ug/ml for all levels

Arabinose 125 ul 250 ul 375 ul 500 ul 25, 50, 75, 100 ug/ml

Rahmnose 50 ul 100 ul 150 ul 200 ul 10, 20, 30, 40 ug/ml

Glactose 125 ul 250 ul 375 ul 500 ul 25, 50, 75, 100 ug/ml

Glucose 250 ul 500 ul 750 ul 1000 ul 50, 100, 150, 200 ug/ml

Xylose 250 ul 500 ul 750 ul 1000 ul 50, 100, 150, 200 ug/ml

72% H2SO4 1724 ul 1724 ul 1724 ul 1724 ul Final concentration 4% H2SO4

Mini-Q water to 50 ml to 50 ml to 50 ml to 50 ml 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…