How Much Hplc Analysis
Usually you make more mobile phase than you need, because you cannot use everything in the bottle (the filter need a few cm of solvent height to avoid air into the system). You also may want to rerun some samples and add a low flow program after the end of the sequence.So for quick estimation: every injection is 1.7 hours, 20 injections is 34 hours.
Count on some delay in the injection, so let's say 36 hours. 36 hours times 0.7 ml/min is 1500 ml of mobile phase. You will use more of A than B.I would make 2000 ml of A and 1000 ml of B and feel safe.If this is more an exam question, you can calculate the exact consumption by plotting the time (min) on the x-axis against the flow (ml/min) of mobile phase A on the y-axis. The consumption of A will be the area under the curve. Some instrument softwares do this calculation automatically (e.g.
Hplc Analysis Method
FIRST VISIT?TRY THESE LINKSBOOK COVER4. Problems with the ChromatogramMany problems in an LC system show up as changes in the chromatogram. Some of these can be solved by changes in the equipment; however, others require modification of the assay procedure. Selecting the proper column type and mobile phase are keys to 'good chromatography.'
Peak Tailing Possible CauseSolution1. Blocked frit1. Reverse flush column (if allowed)b.
Replace inlet fritc. Replace column2. Column void2.
Interfering peak3. Use longer columnb. Change mobile-phase and/or column/selectivity4. Wrong mobile-phase pH4. For basic compounds, a lower pHusually provides more symmetric peaks5. Sample reacting with active sites5.
Add ion pair reagent or volatile basicmodifierb. Change columnB. Peak Fronting Possible CauseSolution1. Low temperature1. Increase column temperature2.
Wrong sample solvent2. Use mobile phase for injection solvent3. Sample overload3. Decrease sample concentration4. Split Peaks Possible CauseSolution1. Contamination on guard or1.
Remove guard column and attemptanalytical column inletanalysisb. Replace guard if necessaryc. If analytical column is obstructed,reverse and flushd. If problem persists, column may befouled with strongly retainedcontaminantse. Use appropriate restoration proceduref. If problem persists, inlet is probablypluggedg. Change frit or replace column2.
Sample solvent incompatible with2. Change solvent; whenever possible,mobile phaseinject samples in mobile phaseD. Distortion of Larger Peaks Possible CauseSolution1. Sample overload1. Reduce sample sizeE. Distortion of Early Peaks Possible CauseSolution1. Wrong injection solvent1.
Reduce injection volumeb. Use weaker injection solventF. Tailing, Early Peaks More Than Later Ones Possible CauseSolution1.
Extra-column effects1. Replumb system (shorter, narrowertubing)b. Use smaller volume detector cellG.
Increased Tailing as k ' Increases Possible CauseSolution1. Secondary retention effects,1. Add triethylamine (basic samples)reversed-phase modeb. Add acetate (acidic samples)c. Add salt or buffer (ionic samples)d. Try a different column2. Secondary retention effects,2.
Add triethylamine (basic compounds)normal-phase modeb. Add acetic acid3. Secondary retention effects,3. Add triethylamine (basic samples)ion-pairH.
Acidic or Basic Peaks Tail Possible CauseSolution1. Inadequate buffering1. Use 50–100 m m buffer concentrationb. Use buffer with pKa equal to pH ofmobile phaseI. Extra Peaks Possible CauseSolution1. Other components in sample1.
Late-eluting peak from previous2. Increase run time or gradient slopeinjectionb. Increase flow rate3. Vacancy or ghost peaks3.
Check purity of mobile phaseb. Use mobile phase as injection solventc. Reduce injection volumeJ. Retention Time Drifts Possible CauseSolution1. Poor temperature control1. Thermostat column2.
Mobile phase changing2. Prevent change (evaporation, reaction,etc.)3. Poor column equilibration3.
Allow more time for column equili-bration between runsK. Abrupt Retention Time Changes Possible CauseSolution1.
Flow rate change1. Reset flow rate2. Air bubble in pump2. Bleed air from pump3. Improper mobile phase3. Replace with proper mobile phaseb. Set proper mobile phase mixture oncontrollerL.
Baseline Drift Possible CauseSolution1. Column temperature fluctuation1. Control column and mobile phase(even small changes cause cyclictemperaturebaseline rise and fall; mostb. Use heat exchanger before detectoroften affects refractive indexand conductivity detectors, orUV detectors at high sensitivityor in direct photometric mode)2. Nonhomogeneous mobile phase2. Use HPLC-grade solvents, high-purity(drift usually to highersalts, and additivesabsorbance, rather than cyclicb. Degas mobile phase before usepattern from temperaturec.
Sparge with helium during usefluctuation)3. Contaminant or air buildup in3. Flush cell with methanol or otherdetector cellstrong solventb. If necessary, clean cell with 1 nHNO 3 (never with HCl)4. Plugged outlet line after detector4. Unplug or replace line(high-pressure cracks cellb.
Refer to detector manual to replacewindow, producing noisy baseline)window5. Mobile-phase mixing problem or5. Correct composition/flow ratechange in flow rateb. To avoid, routinely monitorcomposition and flow rate6. Slow column equilibration,6. Flush with intermediate strengthespecially when changingsolventmobile phaseb. Run 10–20 column volumes of newmobile phase before analysis7.
Mobile phase contaminated,7. Check make-up of mobile phasedeteriorated, or prepared fromb. Use highest grade chemicals and HPLClow-quality materialssolvents8.
Strongly retained materials in8. Use guard columnsample (high k) can elute as veryb. If necessary, flush column with strongbroad peaks and appear to besolvent between injections or periodi-a problemically during analysis9. Mobile phase recycled but9. Reset baselinedetector not adjustedb. Use new mobile phase when dynamicrange of detector is exceeded10.
Detector (UV) not set at10. Change wavelength to UV absorbanceabsorbance maximum but at slopemaximumof curveM. Baseline Noise (Regular) Possible CauseSolution1. Air in mobile phase, detector cell,1. Degas mobile phaseor pumpb. Flush system to remove air fromdetector cell or pump2.
Check system for loose fittingsc. Check pump for leaks, salt build-up,unusual noisesd. Change pump seals if necessary3. Incomplete mobile phase mixing3. Mix mobile phase by hand or use lessviscous solvent4. Temperature effect (column at4.
Reduce differential or add headhigh temperature, detectorexchangerunheated)5. Other electronic equipment on5. Isolate LC, detector, or recorder tosame linedetermine if source of problem isexternal; correct as necessary6.
Pump pulsations6. Incorporate pulse dampener into systemN. Baseline Noise (Irregular) Possible CauseSolution1. Check for loose fittingsc. Check pump for leaks, salt build-up,unusual noisesd.
Change seals if necessarye. Check for detector cell leak2. Mobile phase contaminated,2. Check make-up of mobile phasedeteriorated, or preparedfrom low-quality materials3.
Mobile phase solvents immiscible3. Select and use only miscible solvents4. Detector/recorder electronics4. Isolate detector and recorderelectronicallyb. Refer to instruction manual to correctproblem5. Air trapped in system5. Flush system with strong solvent6.
Air bubbles in detector6. Purge detectorb. Install back-pressure device afterdetector7. Detector cell contaminated (even7. Clean cell by flushing with 1 n HNO 3small amounts of contaminants(never with HCl)can cause noise)8. Weak detector lamp8.
Replace lamp9. Column leaking silica or packing9.
Replace columnmaterial10. Mobile phase mixer inadequate or10. Repair or replace the mixer or mix off-malfunctioningline if isocraticO. Broad Peaks Possible CauseSolution1. Mobile-phase composition changed1. Prepare new mobile phase2.
Mobile-phase flow rate too low2. Adjust flow rate3. Leaks (especially between column3.
Seeand detector)b. Check for loose fittingsc. Check pump for leaks, salt build-up,and unusual noisesd.
Change seals if necessary4. Detector settings incorrect4. Adjust settings5. Extra-column effects:5. Inject smaller column (e.g., 10 µl vs.a.
Column overloaded100 µl) or 1:10 and 1:100 dilutions ofb. Detector response time or cellsamplevolume too largeb. Reduce response time or use smallercellc. Tubing between column andc.
Use as short a piece of 0.007–0.010-detector too long or IDinch ID tubing as practicaltoo larged. Recorder response time tood.
Reduce response timehigh6. Buffer concentration too low6. Increase concentration7.
Guard column contaminated/worn7. Replace guard columnout8. Column contaminated/worn out;8. Replace column with new one of samelow plate numbertypeb. If new column provides symmetricalpeaks, flush old column with strongsolvent9. Void at column inlet9. Open inlet end and fill void or replacecolumn10.
Peak represents two or more10. Change column type to improvepoorly resolved compoundsseparation11. Column temperature too low11. Increase temperature; do not exceed75°C unless higher temperaturesare acceptable to columnmanufacturer12. Detector time constant too large12. Use smaller time constantP.
Loss of Resolution Possible CauseSolution1. Mobile phase contaminated/1. Prepare new mobile phasedeteriorated (causingretention time to change)2. Obstructed guard or analytical2. Remove guard column and attemptcolumnanalysisb. Replace guard if necessaryc. If analytical column is obstructed,reverse and flush; if problempersists, column may be fouledwith strongly retained contaminantsd.
Use appropriate restoration procedure;if problem persists, inlet is probablypluggede. Change frit or replace columnQ. All Peaks Too Small Possible CauseSolution1. Detector attentuation too high1. Reduce attenuation2. Detector time constant too large2.
Use smaller time constant3. Injection size too small3. Use larger sample loop4. Improper recorder connection4.
Use correct connectionR. All Peaks Too large Possible CauseSolution1. Detector attenuation too low1. Use larger attenuation2. Injection size too large2. Use smaller sample loop3. Improper recorder connection3.
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