LCGC 30(12) - LC Resources Inc.
Contents
1. minimizing extra volume To minimize the temperature adjustment require ments a column oven should be used and set to the same temperature as the detector or vice versa so that little or no temperature change is necessary Also be sure to insulate the tubing that connects the column to the detector Some detectors come with insulated connecting tubing but a simple home made insulator can be made by slipping a piece of heavy walled plastic or rubber tubing over the connecting tubing It may take several hours for the detector to warm up and equilibrate with the column temperature so most RI detectors are equipped with a valve that can divert the waste stream back into the mobile phase reservoir In this manner the mobile phase can be recycled and the system can be left with the flow on for several hours to warm up or left pumping continuously so it is always ready to use When samples are run the valve is switched so that the solvent from the detector is directed to waste If you do recycle the mobile phase be sure to replace it once in awhile For a mobile phase that has more than approximately 70 buffer or aqueous component I recommend changing the mobile phase once a week When the mobile phase contains at least 30 organic solvent it can be used longer but it should be replaced every few weeks Mobile phase that is used for too long can gradually change composition because of evaporation of a more volatile component o2. column is sufficient for equilibration It may take longer with the RI detector Watch the baseline www chromatographyonline com when changing the solvent drift is common during solvent changeover so a nondrifting baseline is a good indicator of column equilibration with RI detection As mentioned above be sure to change the mobile phase regularly to avoid problems with microbial growth especially in highly aqueous mobile phases Additional Comments Sometimes RI detectors are used for different applications with either aque ous and nonaqueous solvents When this is the practice be sure to flush the entire system reservoirs degasser pump autosampler and detector with a series of solvents that are mutually miscible For example go from aque ous solvents to 100 acetonitrile or methanol then to organic solvents If you are not sure of the history of the system remove the column and replace it with a piece of capillary tubing Then flush the entire system with 20 30 mL of isopropanol which is miscible with both aqueous and organic solvents Then flush to the desired mobile phase Baseline noise can be a critical fac tor with RI detection Because RI inherently has poor sensitivity when compared to UV or other detectors signal to noise can be a limiting fac tor For this reason you may want to take advantage of larger detector time constants noise filters with RI than with other detectors A good rule of thu
3. in temperature both through use of a thermostated cabinet and a heat exchanger for the incoming solvent However both of these features are compromises between effectiveness and expense The cabinet cannot protect against all environmen tal temperature changes so it may be necessary to take additional action to protect the instrument from local envi ronmental temperature fluctuations Although the laboratory temperature control may be quite good at least as indicated by the thermostat on the wall the local temperature may vary Perhaps a heating vent blows hot or cold air directly at the instrument or direct sunlight may cause local warm ing of the laboratory In some labora tories a different temperature is main tained at night than in the daytime Any of these factors can result in a change in the temperature of the local environment You may need to block a heater vent or redirect it In one labo www chromatographyonline com ratory I visited recently the staff had built a cabinet around the LC system to shield it from local temperature fluc tuations It may be necessary to move the instrument to another location with better temperature control The heat exchanger s job is to change the temperature of the incoming mobile phase to match that of the solvent in the detector cell Because the heat exchanger adds extracolumn dead volume to the system it is a compromise between effi ciency of temperature adjustment and
4. restrictor may cause the permissible cell pressure to be exceeded Because LC systems are operated in a constant flow mode the pressure should be constant This usually is the case but problems with the pumps can cause the pressure to fluctuate suf ficiently that the baseline is disturbed even though other problems such as retention time shifts are not observed Pressure problems because of pump malfunctions often will create cycling baselines To confirm this you can change the flow rate and the frequency of the baseline cycle should change in accordance to the flow rate change For example a change from 1 mL min to 2 mL min should double the frequency of the baseline cycle Com mon sources of pressure fluctuations are faulty check valves leaky pump seals air bubbles in the pump and mote rarely a broken pump piston The easiest things to check are bubbles in the pump and degassing problems make sure the degasser is work ing properly then purge the pump to release any trapped bubbles and resume operation Check valve sonication in methanol for a few minutes often will clean a dirty or sticking check valve or the check valve can be replaced Pump seal replacement is a little more work but is something that can be done by following the instructions in the pump service manual Mobile Phase Problems Any change in the chemical composi tion of the mobile phase will change its refractive index as will the presence o
5. 1032 LCGC NORTH AMERICA VOLUME 30 NUMBER 12 DECEMBER 2012 LC TROUBLESHOOTING Avoiding Refractive Index Detector Problems www chromatographyonline com John W Dolan LC Troubleshooting Editor he refractive index RI detector is unique among common liquid chromatography LC detec tors because it is truly universal in its detection capabilities LC detectors based on the absorbance of ultraviolet UV light are the most popular detec tors because they are simple reliable sensitive and respond to a wide range of sample compounds but only if the analytes have sufficient UV absorbance to detect Fluorescence detectors are much more selective and can be more sensitive but compounds must fluo resce to be detected Mass spectrom etry MS detectors are increasing in popularity and can provide extremely sensitive and selective detection but only if the sample can be ionized RI detectors respond to a universal bulk property of the analyte its refrac tive index Usually referred to as dif ferential refractive index detectors these detectors detect peaks based on the difference in refractive index between the analyte and the background mobile phase This is a benefit that makes the detector universal but also a problem in that the detector also is sensitive to any other factor that affects refractive index The major factors are tem perature pressure and mobile phase composition This month s install
6. al tem perature can be a major problem with RI detectors because the refractive index of a fluid is dependent on its temperature For this reason RI detectors are contained in an insu lated compartment Most commercial detectors can control the temperature above room temperature typically 30 35 C up to 50 60 C although some models can cool the detector as well Also the incoming mobile phase must be at the same tempera ture as the thermostated portion of the detector so heat exchangers are included to stabilize the temperature of the mobile phase Although flow cell volumes are relatively small typi cally 8 10 uL the heat exchanger volume may be 5 10 times this or even more This added volume means that RI detectors usually generate broader peaks than their UV coun terparts with smaller total detector volumes The inherent design and operating principles of RI detectors leave them susceptible to several problem areas Specifically anything that causes changes in the temperature pressure or mobile phase composition will create corresponding changes in the refractive index of the mobile phase as it passes through the sample cell If this is not compensated by the static mobile phase in the reference cell baseline distur bances will occur We ll look at each of these problem areas next Temperature Problems As described above the RI detector is constructed to shield the detector cell from external changes
7. detection of the dust that is left behind Both ELSD and CAD can be operated with gradients which is an additional advantage but they are restricted to mobile phases that are volatile so no phosphate buffer is allowed And if all else fails read the direc tions If you are a normal user of other detectors such as UV fluorescence or MS troubleshooting RI problems may not be second nature Consult the oper ation and service manual for your spe cific detector for troubleshooting and preventive maintenance instructions If you d like advice from other users regarding specific problems consult one of the on line discussion groups such as Chromatography Forum www chromforum org or the HPLC Users Group at LinkedIn www linkedin com Erratum Equation 2 of the October 2012 install ment B Alsehli and JW Dolan LCGC North Amer 10 30 898 902 2012 contained an error and should have read as follows w 4 tg N 3 2 With this change all the calculated values of peak widths and recommended DECEMBER 2012 LCGC NORTH AMERICA VOLUME 30 NUMBER 12 1037 injection volumes should be increased fourfold The discussion and conclusions are still valid For a fully corrected version see www chromatographyonline com Dolan1012 John W Dolan LC Troubleshooting Editor John Dolan has been writing LC Trou bleshooting for LCGC for more than 25 years One of the industry s most respec
8. f dissolved air in the mobile phase For these reasons RI detectors are always operated only in the isocratic not gradient mode and the mobile phase must be thoroughly degassed If you have an in line degasser as is the case for most LC systems today be sure to use it Otherwise helium sparging is suggested to degas the mobile phase Because of the extreme sensitivity of the detector to very small changes in refractive index on line mixing of the mobile phase usually will create problems As a result mobile phases must be hand mixed so that no change in mobile phase composition occurs within the LC system It is best to use the mobile phase as the injection sol vent so the refractive index change at the column dead time is minimized Remember that the RI detector measures the difference in refrac tive index between the contents of the sample and reference cells so the reference cell needs to be purged with fresh mobile phase whenever the mobile phase is changed or replaced with a fresh batch It is a good idea to purge the reference cell daily to ensure its contents are matched with the mobile phase exiting the column When changing from one mobile phase to another or washing the mobile phase into a new column complete equilibration may take longer than you normally allow with UV detection With UV and most other detectors allowing 10 column volumes of mobile phase 15 mL for a 150 mm X 4 6 mm column to pass through the
9. mb is to set the detector time constant at 10 of the peak width at baseline or 20 of the half height width For example if the peak is 10 s wide at the baseline you can use a 1 s time constant A higher time constant value smoothes the baseline but too high a value will smooth off the top of the peaks making them broader and shorter If you are having a hard time distinguishing the source of a base line problem between the pump and mobile phase as opposed to a temperature related problem turn off the pump or set the flow to 0 mL min This will eliminate the pump or mobile phase problem If the base line problem persists it is because of changing temperature www chromatographyonline com Because of its extreme sensitivity to temperature a byword for RI detec tion is patience It will take longer to equilibrate the mobile phase to warm up the detector or settle down from any system change For this reason if time is critical it is prudent to leave the detector turned on and in a mobile phase recycle mode You can reduce the flow rate under these conditions if you desire but this will leave the system in a standby mode that will return rapidly to normal operation If you are looking for alternatives to the RI detector for universal detection consider evaporative light scattering detection ELSD or charged aerosol detection CAD Both of these detec tors rely on evaporation of the mobile phase and then
10. ment describes how RI detectors work and discusses some good practices to follow to get the most out of this powerful detector How It Works Let s first consider how the RI detector works There are specific design differ ences between detectors from different manufacturers but most have the ele ments of the generic detector shown in Figure 1 in common All RI detectors depend on the fundamental property of light s refraction or change of angle as it passes through different materials In the case of the RI detector light passes through the clear walls of the flow cell and through the fluid in the cell With each transition refraction takes place and the direction of the light changes _ Slightly Rather than detect the absolute refractive index which some detectors can most detectors measure the differ ential refraction between a sample flow cell and a static reference cell filled with mobile phase This in effect subtracts the mobile phase background signal from the sample signal Because light of longer wavelengths refracts more than shorter wavelengths a tungsten lamp or light emitting diode LED is used as the light source in most RI detectors In a quick survey I did of commercial RI detectors various manufacturers used light sources producing wavelengths of 660 880 nm After the light has passed through the sample and reference cells it must be detected Most commonly this is done with a pair of photodi
11. odes As the refractive index changes the position of the light beam on the pho todiodes shifts so that more or less light shines on each diode This shift of posi tion can then be detected by comparing the relative intensity of the signal pro duced by the two photodiodes In Fig ure 1 you can see that most of the light strikes the upper diode With a change in refractive index the position of the light beam might move down causing less light to strike the upper diode and more on the lower one The basic components of the RI detector shown in Figure 1 are supplemented in real detectors by 1034 LCGC NORTH AMERICA VOLUME 30 NUMBER 12 DECEMBER 2012 Reference cell Sample cell Dual photodiode Figure 1 Schematic of a generic refractive index detector showing the key components hardware to stabilize the detector and simplify operation The reference cell needs to be filled with mobile phase of the same composition as that fill ing the sample cell without the ana lyte of course To facilitate this a switching valve commonly is included to direct mobile phase through the reference cell to refresh or replace the resident liquid Because it can take several hours for the detector to stabilize the switching valve may be capable of routing the waste line back into the mobile phase reservoir to allow the mobile phase to be recycled during warm up so as to reduce the waste of mobile phase A change in environment
12. r may grow bacteria that can block frits in the system Be sure to replace the reservoir with a clean one instead of refilling the reservoir to prevent passing any con taminants from the previous batch of mobile phase on to the new one 1036 LCGC NORTH AMERICA VOLUME 30 NUMBER 12 DECEMBER 2012 Temperature related problems usually show up as baseline drift Depending on the magnitude of the temperature change and the sensitivity setting on the detector this may be a gradually slop ing or steeply sloping baseline When baseline drift is a problem review the preventive steps listed above and see if there is something you can modify to reduce the problem Pressure Problems A second factor that affects refrac tive index is pressure For the quietest baselines the pressure in the flow cell needs to be constant Most RI flow cells have an upper pressure limit of no more than approximately 100 psi 7 bar and the use of a back pressure restrictor after the cell is common A back pressure restrictor can be thought of as a spring loaded check valve that maintains a fixed pressure such as 75 psi 5 bar at all times This will keep the pressure constant and also will keep the system from exceeding the maximum cell pressure A piece of cap illary tubing after the flow cell also can function as a back pressure restrictor but the pressure will be related to the flow rate if the flow rate is inadver tently set too high a capillary
13. ted profes sionals John is currently the Vice President of and a principal instructor for LC Resources Walnut Creek California He is also a member of LCGC s editorial advisory board Direct correspondence about this column via e mail to John Dolan LCResources com For more information on this topic please visit www chromatographyonline com Dolan
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