Agilent Dimension Software for ELSD
Contents
1. Figure 2 Configuration of HPLC system for direct injection of a single compound It is recommended that repeat injections be made at 10 time intervals across the gradient such that for a 10 min run injections every 1 min should be 2 10 Calibration Procedure made Using these conditions a chromatogram similar to that shown in Figure 3 on page 10 should be obtained 400 ELSD Response across a Gradient using Fixed Evaporator Gas 5 Fluorocytosine Agilent 38X ELSD 5 FC Peak Area 350 ELSD mV UV 254 nm 5 FC Peak Area UV mAu Figure 3 4 6 Time mins 800 700 1000 800 600 400 200 SAW eary Jead 94 G s nyw ealy Jead 94 9 Direct injections of single component across a solvent gradient with UV data Agilent Dimension Software for ELSD User Manual Calibration Procedure 2 Implementing Real Time Gas Control The Agilent 38x ELS Detector can store in memory a series of time based events within a single timetable This timetable allows the operational settings of the ELSD to be changed in real time during a run The timetable can be used to program the gas flow in order to compensate for the change in ELSD response across a solvent gradient Alternative2. ELSD in order to overcome any solvent enhancements effects to deliver a uniform response when running gradients 6 Agilent Dimension Software for ELSD User Manual Agilent Dimension Software for ELSD User Manual ee 2 Calibration Procedure Evaluating Solvent Enhancement 8 Correcting Solvent Enhancement 9 Implementing Real Time Gas Control 11 In order to compensate for the solvent enhancement effect on the Agilent 38x ELSD a gas timetable must be developed and programmed into the detector To achieve the appropriate gas flow timetable for the solvent gradient the following steps must be taken 1 2 3 4 5 Inject sample under typical LC gradient conditions Characterise ELSD gradient response for single compound under typical LC conditions Use Agilent 38x ELSD Dimension software to formulate gas flow program Download Gas program to ELS Detector Inject sample using real time gas control program These procedures are described in more detail below with real chromatographic examples ott Agilent Technologies 2 Calibration Procedure Evaluating Solvent Enhancement LC Pump A S LC Column Configure the HPLC system according to the required analytical protocol An example of the typical hardware configuration is shown in Figure 1 on page 8 Under the required analytical conditions inject a sample or standard mixture to obtain a reference chromatogram under typical LC conditions This pro
3. 93 10 8 2 30 1 30 3 0 43 2 03 7 07 13 6 2 89 1 89 3 0 63 2 23 8 22 17 4 3 70 2 70 3 0 90 2 50 9 38 19 1 4 06 3 06 3 1 02 2 62 10 53 18 6 3 96 2 96 3 0 99 2 59 For configuration of HPLC system for direct injection of a single compound see Figure 2 on page 9 1 Predicted Linear Gas Flow Increment D Change in Response at time x C Change in Re sponse at Injection 1 C e g at time 1 44 Predicted Linear Gas Flow Increment 1 09 1 00 0 09 tor E e g at time 2 54 Predicted Log Gas Flow Increment 0 47 3 0 16 Predicted Log Gas Flow Increment F Predicted Linear Gas Flow Increment D Correction Fac Estimated Gas Flow Required G Initial Gas Flow at injection 1 Predicted Log Gas Flow Incre ment at time x D e g at time 4 77 Estimated Gas Flow Required 1 60 0 36 1 96 This gas flow program is a step wise gradient and is only a guide to the required gas flow required to achieve uniformity The next step is to input these values into Dimension software which will interpolate between the set values and create a smooth gas transition across the gradient Agilent Dimension Software for ELSD User Manual Calibration Procedure 2 Implementing Real lime Gas Contro Using Dimension software to develop gas program The start and end values for the gas can be inputted into Dimension to give a smooth gas profile as shown in Figure 4 on page 15 See the Dimension on line help for further inform
4. Agilent Dimension Software for ELSD User Manual Agilent Technologies Notices Agilent Technologies Inc 2011 No part of this manual may be reproduced in any form or by any means including elec tronic storage and retrieval or translation into a foreign language without prior agree ment and written consent from Agilent Technologies Inc as governed by United States and international copyright laws Manual Part Number 64260 90320 Edition 06 11 Printed in Germany Agilent Technologies Hewlett Packard Strasse 8 76337 Waldbronn This product may be used as a com ponent of an in vitro diagnostic sys tem if the system is registered with the appropriate authorities and com plies with the relevant regulations Otherwise it is intended only for gen eral laboratory use Warranty The material contained in this docu ment is provided as is and is sub ject to being changed without notice in future editions Further to the max imum extent permitted by applicable law Agilent disclaims all warranties either express or implied with regard to this manual and any information contained herein including but not limited to the implied warranties of merchantability and fitness for a par ticular purpose Agilent shall not be liable for errors or for incidental or consequential damages in connection with the furnishing use or perfor mance of this document or of any information contained herein Sho
5. ation on using Dimension Agilent ELSD Dimension File Edit Instrument Help SH SA amp x Glcomi yl Timetable Properties Time Mins Evap Gas SLM Function PMT Gain Smoothing v Gas FR Evap Temp Led PMT Gain Evap Gas SLM 4 6 5 Time Mins Figure 4 Gas program developed using Dimension software Gas program from Table 2 on page 14 Having developed the initial gas profile the program is downloaded to the ELSD and the direct injections of the single compound are repeated to assess the effect of the gas control Figure 4 on page 15 shows the result of using the gas flow program in Figure 4 on page 15 compared to the result obtained without gas flow control as shown in Figure 3 on page 10 Agilent Dimension Software for ELSD User Manual 15 2 16 Calibration Procedure Agilent ELSD with real time control a i a Qa o ao N oO D fo nn a 2 D o oO o a 3 z a a g a I ror a E rA K o 5 oO D 2 3 gt gt gt w Figure 5 Re injection of single compound using real time gas control program See Figure 2 on page 9 for comparison If the initial gas flow program is insufficient to control the changing peak response during the gradient then Dimension software can be altered accordingly i e by applying a different correction factor and the new program reloaded onto the ELSD Once this itera
6. in compound response across a solvent gradient is a consequence of the viscosity difference between mobile phase A and B During a typical reverse phase gradient the viscosity decreases as the proportion of organic mobile phase increases This transition of decreasing solvent viscosity affects the nebulisation process of the ELSD causing an increase in the number of droplets formed which subsequently leads to an enhancement in the detector s response This non uniform response across a solvent gradient has several implications when quantifying with aerosol based detectors 1 A slight shift in a compound s retention time between injections will lead to poor reproducibility in area response 2 Irreproducible gradient programs will produce poor accuracy due to variations in solvent composition If quantifying unknown compounds where calibration standards don t exist using chemically dissimilar calibration standards then this enhancement effect of the mobile phase has to be considered otherwise large errors are observed The Agilent 38x ELSD has the ability to compensate for this solvent enhancement by controlling the number of particles entering the detection stage in real time during the gradient The detector uses an on board timetable that can be programmed to control the detector s evaporation gas flow ee Agilent Technologies 5 1 Introduction In This Book This guide outlines how to calibrate amp program the Agilent 38x
7. ly the timetable can be customized to create adjustable LED intensities across a sample injection for mixtures containing samples with wide concentration ranges The single timetable stored on board the ELSD is only customizable using the Agilent 38x ELSD DIMENSION software which is a standalone software application NB Once a timetable has been downloaded to the instrument it cannot be removed Therefore if a timetable of events is no longer required either the trigger cable should be disconnected to prevent the timetable from being initiated again or a blank timetable with constant detector settings programmed should be downloaded to the ELSD instead This section describes how to use Dimension software to formulate a real time gas program Creating a real time program In order to create or modify the on board timetable DIMENSION software must be installed on your PC The DIMENSION software package allows you to create a timetable on a PC which can subsequently be downloaded to the ELSD for later use The ELSD can only store a single timetable in memory so the DIMENSION software can be used to create amp save multiple timetables that can be downloaded individually at a later date For further information on how to use DIMENSION software please refer to the DIMENSION software on line help Agilent Dimension Software for ELSD User Manual 11 2 12 Calibration Procedure Calculating the shape of the gas g
8. r formed or adhered to could result in damage to the product or loss of important data Do not proceed beyond a CAUTION notice until the indicated condi tions are fully understood and met A WARNING notice denotes a hazard It calls attention to an operating procedure practice or the like that if not correctly performed or adhered to could result in personal injury or death Do not proceed beyond a WARNING notice until the indi cated conditions are fully under stood and met Agilent Dimension Software for ELSD User Manual In This Book In This Book This manual contains information on the Agilent Dimension Software for ELSD Agilent Dimension Software for ELSD User Manual Contents Contents 1 Introduction 5 2 Calibration Procedure 7 Evaluating Solvent Enhancement 8 Correcting Solvent Enhancement 9 Implementing Real Time Gas Control 11 4 Agilent Dimension Software for ELSD User Manual Agilent Dimension Software for ELSD User Manual eece 1 Introduction A well known property of aerosol based detectors is their response dependency on mobile phase composition For example when using a reverse phase gradient the response of analytes eluting at the end of the gradient will be larger than those at the beginning of the gradient For compounds of equal concentration the response of late eluting compounds can be 2 8 x larger than those at the early part of the gradient This enhancement
9. radient From the data obtained in Figure 3 on page 10 whereby the gradient response profile was characterized with a single component a gas gradient can be defined Using the peak area data from the single component injections the values must be normalized against the first injection to determine the change in signal response These data are shown in Table 1 on page 12 Table 1 Data for single compound injection A B c D Peak Injection Peak Area Change in Retention No mV min response Time from mins injection 1 0 29 1 4 7 1 00 1 44 2 5 1 1 09 2 54 3 6 9 1 47 3 68 4 7 9 1 68 4 77 5 9 8 2 09 5 93 6 10 8 2 30 7 07 7 13 6 2 89 8 22 8 17 4 3 70 9 38 9 19 1 4 06 10 53 10 18 6 3 96 For direct injections of single component across a solvent gradient with UV data see Figure 3 on page 10 i Change in response from injection 1 D Response at time x C Response at injection 1 C e g at time 7 07 Change in response from injection 1 13 6 4 7 2 89 By calculating the gradual increase in peak height of the single component the relative change in ELSD response across the gradient can be assessed From Agilent Dimension Software for ELSD User Manual Calibration Procedure 2 the data in Table 1 on page 12 the ELSD response changes by a factor of 4 across the 10 min solvent gradient Calculating the required gas flow from the data in Table 1 on page 12 requires an experimental trial and error proced
10. tive process of editing and optimising the gas program has achieved the desired uniformity for the single compound injections then the HPLC can be re configured back to its original state as shown in Figure 1 on page 8 The ELSD stores the gas program in its memory and it is triggered by an input signal from the auto sampler To implement this start signal the ELSD remote signal cable must be connected from the I O connector on the rear of the unit to the A S trigger cable Agilent Dimension Software for ELSD User Manual Calibration Procedure 2 Implementing Real Time Gas Control Agilent Dimension Software for ELSD User Manual 17 www agilent com In This Book The manual describes the following Introduction e Calibration Procedure e Evaluating Solvent Enhancement Correcting Solvent Enhancement e Implementing Real Time Gas Control Agilent Technologies 2011 Printed in Germany 06 11 G4260 90320 EE Agilent Technologies
11. uld Agilent and the user have a separate written agreement with warranty terms covering the material in this document that conflict with these terms the warranty terms in the sep arate agreement shall control Technology Licenses The hardware and or software described in this document are furnished under a license and may be used or copied only in accor dance with the terms of such license Restricted Rights Legend If software is for use in the performance of a U S Government prime contract or subcon tract Software is delivered and licensed as Commercial computer software as defined in DFAR 252 227 7014 June 1995 or as a commercial item as defined in FAR 2 101 a or as Restricted computer soft ware as defined in FAR 52 227 19 June 1987 or any equivalent agency regulation or contract clause Use duplication or dis closure of Software is subject to Agilent Technologies standard commercial license terms and non DOD Departments and Agencies of the U S Government will receive no greater than Restricted Rights as defined in FAR 52 227 19 c 1 2 June 1987 U S Government users will receive no greater than Limited Rights as defined in FAR 52 227 14 June 1987 or DFAR 252 227 7015 b 2 November 1995 as applicable in any technical data Safety Notices CAUTION A CAUTION notice denotes a hazard It calls attention to an operating procedure practice or the like that if not correctly pe
12. ure but a very close estimate of the gas flow program to start with can be calculated This calculation of gas flow value at each 1 min step across the gradient is shown in Table 2 on page 14 By calculating the change in ELSD response at each minute across the gradient normalized against the first injection a value for the necessary increase in gas can be determined However the effect of increasing gas flow does not have a linear relationship on the ELSD response because of the logarithmic response of the ELSD Therefore to compensate for this non linear relationship a correction factor must be applied By dividing the predicted linear gas increment by the correction factor E i e 3 then a more realistic gas increase increment is obtained From this logarithmic gas increment value the estimated gas flow across the gradient can be made as highlighted in red within Table 2 on page 14 Agilent Dimension Software for ELSD User Manual 13 2 14 Calibration Procedure Table 2 Data for single compound injection A B c D E F G Peak Single Changein Predicted Correction Predicted Estimated Retention Compound Response Gas flow Factor due Gas Gas Flow Time Peak Area vsfirstinj increment to ELSD ilog Increment Required mins mV min if linear response log resp 0 29 4 7 1 00 1 00 3 1 1 60 1 44 5 1 1 09 0 09 3 0 03 1 63 2 54 6 9 1 47 0 47 3 0 16 1 76 3 68 7 9 1 68 0 68 3 0 23 1 83 4 77 9 8 2 09 1 09 3 0 36 1 96 5
13. vides a useful control when comparing with the data obtained under gas flow control 10T 0 0000 0000 0 0000 Figure 1 HPLC Configuration under typical operating conditions Agilent Dimension Software for ELSD User Manual Calibration Procedure 2 Correcting Solvent Enhancement ELSD LC Pump LC Column A S Agilent Dimension Software for ELSD User Manual Having established a response profile for the sample mixture the contribution of solvent enhancement must be determined by calibrating the solvent gradient using a single compound The single compound is injected directly into the ELSD under the same gradient conditions to achieve a response profile The HPLC solvent must be re configured for this test to allow direct injections to be made into the ELSD The compound that elutes at the mid point of the gradient should be used to characterize the solvent gradient In order to make direct injections into the ELSD but maintain the same dwell volume for the gradient program the LC system must be configured as shown in Figure 2 on page 9 The HPLC column must be connected between the HPLC pump and auto sampler and the auto sampler must be connected directly to the ELSD
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