TN 700: Optimizing the ASI-100 Autosampler Injection
Contents
1. ZY DIONEX Technical Note 700 Optimizing the ASI 100 Autosampler Injection Precision and Linearity Using PrimeSyringe INTRODUCTION When laboratories need to quantify analytes at high precision an autosampler with a precise mechanic and fluidic design is mandatory However even with quality instruments users need to follow basic rules of proper Operation to achieve good day in and day out perfor mance There are several characteristics of automated injectors that a chromatographer should be aware of The most common instrument related causes for poor quantitative precision are e Gas bubbles in the injection system e Inappropriate draw speed the speed at which the autosampler draws the sample into the injection loop e Worn out parts needleport seal rotor seal syringe plunger etc e Insufficient vial pressure equilibration during sample loading This technical note focuses on the effects of gas bubbles and draw speed on autosampler performance using the Dionex ASI 100 Autosampler The instrument s user manual addresses the two other causes of substandard autosampler performance mentioned above This technical note shows how an automated gas bubble removal command PrimeS yringe effectively eliminates gas bubbles and therefore significantly improves autosampler performance Now sold under the Thermo Scientific brand Thermo SCIENTIFIC ease of use and reliability This document describes the PrimeSyringe pro2. 0161 43 43 03 Switzerland 062 205 99 66 United Kingdom 01276 691722 94088 3603 84119 1484 Atlanta GA 770 432 8100 Designed developed and manufactured under an NSAI registered ISO 9001 Quality System 408 737 0700 801 972 9292 Marlton NJ 856 596 06009 LPN 1646 PDF 10 04 2004 Dionex Corporation
3. capillary 13 m x 0 13 mm i d Flow Rate 1 0 mL min Inj Volume 5 10 20 40 and 80 uL faves Sample Concentration 10 mg L caffeine Vial 2 mL crimped vial with unslotted septa Detection Wavelength 272 nm Column Temp 25 C Sample Number Draw Speed 25 uL s Figure 5 Test 1 ASI 100 with a draw speed of 25 uL s three consecutive injections per vial 30 uL gas bubble in the syringe Delay Time 5s trend plot n 9 1 9 RSD obtained Instruments P680A LPG ASI 100T TCC 100 and PDA 100 Peak Area UV_VIS_1 THE EFFECT OF GAS BUBBLES AND SAMPLE DRAW SPEED Peak Area Precision The precision of peak areas depends not only on the autosampler s capability to inject samples precisely but also on the precision of data processing and absence of Sample Number application specific effects e g analyte adsorption to Figure 6 Test 2 ASI 100 with a draw speed of 25 uL s three consecutive injections per vial after PrimeSyringe trend plot minimized through the experimental setup peak area n 9 0 40 RSD obtained fluidic surfaces However when all other effects are precision is a good measure of the autosampler s injection volume precision The following application example was an actual faite support case from a Dionex customer The customer observed poor peak area precision with relative standard deviations in the range of 1 2 Troubleshooting the customer s instrument we found that th
4. cedure for purging gas out of the system and recommends draw speeds for common syringe sizes The described operations help achieve typical peak area relative standard deviations RSDs of better than 0 2 and a high injection linearity This performance is demonstrated using two application examples Operating the ASI 100 with Chromeleon software allows users to automate wash routines and integrate them into their daily analytical work EXPERIMENTAL PrimeSyringe Procedure The PrimeSyringe command automatically elimi nates gas bubbles from the autosampler flow path and therefore improves performance ease of use and reliability The operational principle of PrimeSyringe is based on preconditioning the injection syringe with a wash liquid and then eluent prior to sample injection During this procedure all gas bubbles are also purged We recommend performing the PrimeSyringe procedure with isopropanol after which the procedure must be repeated with the current eluent To avoid carryover this technique is strictly forbidden using samples Steps 1 3 of the following procedure are executed automatically without user interaction Step I The pump flow is off The injection valve switches to the Load position allowing pump flow to bypass the sample loop and needle The needle descends into the selected wash vial and the syringe draws a full syringe volume Figure 1 Waste c MN _ Injection syringe gt Wash
5. e poor precision could be attributed to gas in the injection system Gas bubbles in the injection system are a common issue with autosamplers Some designs attempt to Sample Number resolve the gas issue by using a flow through injection Figure 7 Test 3 ASI 100 with a draw speed of 10 uL s three oe consecutive injections per vial no gas bubble trend plot n 9 side effect of a significantly increased and variable 0 20 RSD obtained syringe Unfortunately this design has the undesirable dead volume Gas bubbles originating from incompletely degassed eluents or small leaks may form in the injection system for example when the instrument is not operated over a weekend As shown in our example an automated syringe priming procedure eliminates the issue Technical Note 700 5 A gas bubble in the syringe causes a dramatically negative effect on the peak area RSD of the peak area see Test 1 in Figure 5 The automatic PrimeSyringe procedure effectively removes the gas bubble and brings the relative standard deviation back into an acceptable range customer s target was less than 0 5 RSD Although the draw process of the sample took only 0 2 s with the 25 uL s draw speed setting satisfactory RSD values were obtained because there was no gas bubble in the syringe see Test 2 in Figure 6 Reducing the draw speed to 10 uL s Test 3 improves the RSD further Figures 6 and 7 compare the results These results were stat
6. ion Example Sample Eluent Concentration Component A 0 02 M tetrabutylammonium sulfate in Vials HPLC grade water pH 2 3 Detection Eluent Wavelength Component B Acetonitrile Column Temp Composition Eluent A B 40 60 isocratic premixed Draw Speed Column Dionex Acclaim 120 C18 5 um 150 x 4 6 mm Delay Time Flow Rate 0 8 mL min Instruments Inj Volume 5 uL 4 Sample Position Injection Volume 10 00 pl 15 x 18 x Type nia Sample Ho Connected Ea Retention Time Sample End inet e Ready Busy Advanced Script Button Properties Br xj inject Wait k General Autosize Button Command sid Mode Miscellaneous i S ampler PrimeSpringe Check Stop Mode Bungie E Ty ItNow Beset Wash wash Settings Prime Syringe a External MSV Relays and Inputs ___ sfmag DEMO oK 7 0 5 g L chloranil dissolved in acetonitrile 2 mL crimped vials with unslotted septa 289 nm 25 C 25 uL s for Test 1 and 2 and 10 uL s for Test 3 5s P580A HPG 2 with external degasser ASI 100T STH 585 and UVD 340U Optimizing the ASI 100 Autosampler Injection Precision and Linearity Using PrimeSyringe Autosampler Injection Linearity A peak area trend plot is presented for each test Fluent HPLC grade water setup The customer s target was to achieve an RSD of less than 0 5 The 2S limits of the trend plot have been set accordingly Backpressure Coil PEEK restriction
7. istically confirmed performing an F test and t test at 95 confidence level AUTOSAMPLER INJECTION LINEARITY Modern autosamplers allow the injection of variable volumes Therefore it is important to ensure an injec tion linearity range for the syringe used During the ASI 100 Operational Qualification OQ linearity test procedure the autosampler injects five different vol umes 5 80 uL of the same caffeine standard The injection volume and the caffeine peak area are pre sented graphically The correlation coefficient r of the regression line is expressed in percent and the RSD of the residuals is calculated The acceptance criteria are correlation coefficients r 99 99 and RSD lt 0 5 In this technical note we present a case where the ASI 100 OQ linearity test initially failed but passed after performing PrimeSyringe The ASI 100 syringe was visually inspected for absence of gas bubbles prior to starting both OQ tests EEYDIONEX lt ON THE WEB Table 2 ASI 100 Linearity Test Correlation Coefficients Value before PrimeSyringe 99 99700 0 91 Value after PrimeSyringe 99 99991 0 15 Limits 99 99000 0 50 Table 2 summarizes the results of the two linearity tests The first test passed the correlation coefficient limit r 99 997 limit 99 99 but did not pass the second specification RSD 0 91 limit lt 0 5 The repeated ASI 100 linearity test after PrimeSyringe was performed not on
8. ly passed the RSD specification RSD 0 15 but also improved the correlation coefficient value r 99 99991 CONCLUSIONS The ASI 100 PrimeSyringe procedure increases confidence in your chromatographic results It is available for Chromeleon 6 30 SP 8 6 40 SP6 6 50 SP4 and all higher Chromeleon versions The autosampler s liquid flow path is kept bubble free ensuring high chromatographic peak area reproducibility The described procedure runs fully automatically Thus it is user independent and can be integrated into a sequence Use isopropanol to remove air bubbles and repeat the PrimeSyringe procedure with the appropriate eluent composition 1 e starting eluent composition The two application examples clearly demonstrate the benefit of the PrimeSyringe procedure ASI 100 is a trademark and Acclaim and Chromeleon are registered trademark of Dionex Corporation Dionex Corporation Dionex Corporation Dionex U S Regional Offices Dionex International Subsidiaries 1228 Titan Way Salt Lake City Technical Center Sunnyvale CA 408 737 8522 Australia 61 2 9420 5233 Austria 01 616 51 25 Belgium 03 353 42 94 Canada 905 844 9650 China 852 2428 3282 P O Box 3603 1515 West 2200 South Suite A Westmont IL 630 789 3660 Denmark 36 36 90 90 France 01 39 30 0110 Germany 06126 991 0 Italy 06 66 51 50 52 Japan 06 6885 1213 Sunnyvale CA Salt Lake City UT Houston TX 281 847 5652 Korea 82 2 2653 2580 The Netherlands
9. ve gas bubbles from a 1000 or 2500 uL syringe With either large syringe a full syringe injection of eluent is usually sufficient to remove gas bubbles In addition to effectively removing gas bubbles the PrimeSyringe command allows for a complete liquid exchange in the autosampler As can be seen in Figure 3 just switching between Load and Inject does not exchange the liquid between the syringe and switching valve To completely exchange the liquid in the flow path of the autosampler the PrimeSyringe procedure has to be repeated with the current eluent Therefore the PrimeSyringe procedure may also be used when switching between incompatible applications e g from reversed phase to normal phase applications The PrimeSyringe command can easily be executed from the ASI 100 Chromeleon control panel available on every Chromeleon installation CD as illustrated in Figure 4 The PrimeSyringe button can be included easily in any existing control panel Technical Note 700 3 Chromeleon ASI 100 of DEMO System on CSFMAG A File Edit Control Batch W Sample Information View Workspace Qualification Inject Control Datasource Sequence Current Sample Wellness Temperature 20 C Set Temperature OK Abbrechen For Help press F1 Figure 4 ASI 100 Chromeleon control panel with PrimeSyringe command Peak Area Precis
10. vial Column Figure 1 Needle in the wash vial and syringe draws full volume Step 2 The injection valve switches back to the Inject position while the needle remains in the wash vial The syringe plunger pushes the liquid contained in the syringe to waste Figure 2 Sample Waste needle o C00 Injection syringe Needle port Wash vial Column Figure 2 Injection valve in the Inject position with the needle still in the vial Step 3 The injection valve switches to Load position and the procedure steps 1 and 2 is repeated four more times During this process the wash liquid reaches the syringe removing gas bubbles and exchanging the syringe contents After completion of the PrimeSyringe procedure the needle moves back to the needle port and the injection valve switches to Inject After reestablishing the pump flow the flow path is as illustrated in Figure 3 Although most of the liquid in the injection system is replaced by the current eluent the segment between the syringe and the valve is not it remains filled with the wash period Sample Waste needle J Injection syringe Needle port Wash vial Column Figure 3 ASI 100 liquid flow path after PrimeSyringe procedure with isopropanol Next the PrimeSyringe procedure is repeated with the current eluent composition of the application though without modifiers such as buffer salts to ensure that the whole liquid flo
11. w path of the autosampler is purged with the same liquid Observing this procedure more closely reveals that a few parameters must be considered prior to starting PrimeSyringe 2 Optimizing the ASI 100 Autosampler Injection Precision and Linearity Using PrimeSyringe e The pump flow must be off If not the pump would deliver eluent into the wash vial as soon as the injection valve switches from Load to Inject see Figure 2 e The syringe wash and dispense speed have to be set according to the syringe used see Table 1 Note that wash speed instead of draw speed has to be set when using a wash vial e A PrimeSyringe wash solution and the wash vial position have to be defined e The procedure has to be run twice first with isopropanol then with the current eluent We recommend the use of isopropanol 2 propanol as wash solution as it effectively removes gas bubbles from the system The PrimeSyringe procedure has to be repeated with the current eluent to remove isopropanol completely from the system In case the current eluent contains a buffer a corresponding eluent composition without buffer addition is recommended to avoid crystallization or microbiological growth in the syringe Table 1 lists the recommended ASI 100 settings for different syringes Table 1 Recommended ASI 100 Settings for PrimeSyringe ran ed Dispense speed uL s 50 aye 9 In general PrimeSyringe is not required to remo
Download Pdf Manuals
Related Search