We describe a month prospective study in a clinical diagnostic laboratory in which we evaluated the imprecision of HPLC retention times and determined the retention times for hemoglobin variants seen in a multiethnic setting. We compared the imprecision of retention time with the imprecision of retention time normalized to the retention time of hemoglobin A 0 HbA 0 and to the retention time of HbA 2. Alkaline and acid hemoglobin electrophoresis, and in certain cases globin chain electrophoresis, isoelectric focusing, and DNA analysis, were performed to document the identities of the hemoglobin variants.
Among samples tested, we encountered 34 unique hemoglobin variants and 2 tetramers. Eighteen variants and 2 tetramers could be identified solely by retention time and 3 variants by retention time and proportion of total hemoglobin.
Four variants could be identified by retention time and peak characteristics and eight variants by retention time and electrophoretic mobility. Retention time on HPLC was superior to electrophoresis for the differentiation and identification of six members of the HbJ family, four members of the HbD family, and three variants with electrophoretic mobilities identical or similar to that of HbC. Six variants with electrophoretic mobilities identical or similar to that of HbS could be differentiated and identified by retention time and proportion of total hemoglobin.
Conclusions: The retention time on HPLC is reliable, reproducible, and in many cases superior to conventional hemoglobin electrophoresis for the detection and identification of hemoglobin variants.
Confirmatory testing by electrophoresis can be eliminated in the majority of cases by use of retention time, proportion of total hemoglobin, and peak characteristics of HPLC. The laboratory diagnosis of hemoglobinopathies and thalassemias is of growing importance, particularly because of an increasing requirement for antenatal diagnosis of significant disorders of globin chain synthesis. Family studies can be of considerable importance in elucidating the nature of disorders of hemoglobin synthesis, but definite identification can be achieved only by DNA analysis or amino acid sequencing 1 2 3.
Alkaline and acid hemoglobin electrophoresis are the most widely used methods for investigating hemoglobin variants and hemoglobinopathy. In addition, there are other variants with electrophoretic mobilities identical or similar to those of HbS and HbC.
Consequently, acid electrophoresis is needed for the identification of the aforementioned variants. Hemoglobin fraction analysis by cation-exchange HPLC has the advantage of quantifying HbF and HbA 2 along with hemoglobin variant screening in a single, highly reproducible system, making it an excellent technology to screen for hemoglobin variants and hemoglobinopathies along with the thalassemias 14 5 6 7.
The simplicity of the automated system with internal sample preparation, superior resolution, rapid assay time, and accurate quantification of hemoglobin fractions makes this an ideal methodology for the routine clinical laboratory 5 6. Numerous automated HPLC systems are now commercially available, and evaluations have been published 8 9 10 Much of the published literature on the use of HPLC for the investigation of hemoglobinopathies and thalassemias has evaluated its effectiveness in newborn-screening programs 13 14 15 We report here the results for HPLC performance in a large prospective study of samples over a month period in a multiethnic population.
The Variant II dual pumps deliver a programmed buffer gradient of increasing ionic strength to the cartridge, where the hemoglobin fractions are separated based on their ionic interaction with the cartridge material.
The separated hemoglobin fractions pass through a flow cell, where absorbance is measured at nm; background noise is reduced with the use of a secondary wavelength at nm. The integrated peaks are assigned to manufacturer-defined windows derived from the retention time, i. If a peak elutes at an retention time not predefined, it is labeled as an unknown.
Over a month period, samples were analyzed in the Special Hematology Laboratory at Bellevue Hospital Center for quantification of hemoglobin fractions and screening for hemoglobin variants.
For specimens that showed chromatogram patterns consistent with sickle trait, the presence of HbS was confirmed by use of the sodium metabisulfite reduction test The presence of HbH was confirmed by use of the brilliant cresyl blue test for inclusion bodies Certain specimens were forwarded to the Mayo Medical Laboratories Rochester, MN for confirmation, where additional testing was performed by isoelectric focusing, globin chain electrophoresis, and unstable hemoglobin screen.
Amino acid sequencing was performed on selected specimens.Mar 14 Read Jan 28 Read Apr 01 Read Mar 30 Read Mar 22 Read Feb 29 Read Feb 12 Read Jun 29 Read Oct 22 Read Jul 30 Read Feb 14 Read Jul 22 Read Apr 03 Read May 20 Read The aim in high performance liquid chromatography HPLC and gas chromatography GC is to get good peak shape and good separation.
The most desirable outcome is the generation of symmetric peaks. Peak fronting is the name given to asymmetric peaks having a wider front half of the peak compared to the back half.
HPLC troubleshooting Guide
A quantitative peak description imagines a peak divided vertically into two halves, from the apex of the peak, vertically down to the baseline. Acceptable peaks have values 0. A peak has unacceptable peak fronting if the tailing factor is less than 0.
Other industries use a similar quantitative measure known as the asymmetric factor, A S. Peaks fronting occurs when the sample capacity of the analytical column is exceeded, which can happen in both GC and HPLC experiments.
In GC separations, this can lead to anti-Langmuir behaviour — with the distribution of the solute being non-idealized between the mobile and stationary phases — and leads to peak fronting. At a fixed temperature, any given solute will have a maximum vapour pressure specific to that temperature. The solute can evaporate if the vapour pressure of the solute is below the maximum.
Chromatography / Hints and tips / HPLC troubleshooting
If a large sample volume is injected, the maximum vapour pressure may be reached when no more solute can evaporate. The solute that cannot evaporate remains in the stationary phase until the vapour pressure drops.
It appears that the stationary phase is overloaded compared to the mobile phase. Another cause of peak fronting is channelling in a packed column.
In this case, a narrow channel forms in the stationary phase, sometimes as a result of air bubbles being pushed through. The channels allow some of the solute molecules to get in front of the main body of solute.
They can elute slightly in front of the main peak, leading to peak fronting.A chromatographer always looks forward to getting perfect shaped peaks for each and every analysis but in reality peaks get distorted due to numerous reasons.
Distortions are frustrating but if proper corrective steps are taken peak shape distortions can be avoided. A flat top peak response arises when the detector gets overloaded with the sample. In such case detector sensitivity is much higher and it gets saturated to give a broad flat top peak. One option is to attenuate the signal response not a practical solution as detection of other peaks will also get affected.
A more viable option is to reduce the concentration of sample or volume of injection. Peak tailing is a common distortion arising due to ionization of surface silanol groups to — Si-O — which provide cationic exchange sites. The solution lies in use of high purity grade silica support along with a selection of the recommended pH range Use of buffer control additives also limits ionization of silanol groups on the silica surface.
Additives such as Trifluoroacetic acid TFA an ion pairing agent helps suppress ionization. Buffer additives in the concentration range 10— 25 mM are sufficient for most applications. Frit blockage or void formation can be prevented by filtering mobile phase solvents, use of inlet filters and replacement of pump seals before they begin to wear and release garbage particles. Low purity silica often contains metal impurities which promote ionization of silanol groups.
Use of high purity silica stationary phase helps in tailing due to chelation with metal ion in stationary phase. Generally peak fronting as a result of channeling inside the column.
It is best to replace the column or otherwise operate within the recommended pH limits.
What's the reason for broad peaks, peak tailing, or peaks fronting?
Column overload can also result in peak fronting. Use dilute sample or reduce the sample injection volume. Incompatibility of mobile phase with sample often results in peak fronting. Dissolve sample in mobile phase or another compatible solvent. Shoulder peaks and split peaks often result due to presence of two closely unresolved compounds.
Reduce sample size or use a diluted solution often resolves the split. Splitting off peaks is also caused by frit blockage. Reverse flow with 20 — 30 ml of mobile phase often resolves the peak splits. When split is caused due to presence of two closely eluting compounds use sample cleanup prior to injection.
As you have seen above a number of causes of peak shape distortions are common such as pH control of mobile phase, blockage due to particulate contamination, blocked frits and column overload. The shape of the distortions can be improved by taking corrective steps and other recommended steps whenever necessary. Dr Deepak Bhanot is a seasoned professional having nearly 30 years expertise beginning from sales and product support of analytical instruments.
His mission is to develop training programs on analytical techniques and share his experiences with broad spectrum of users ranging from professionals engaged in analytical development and research as well as young enthusiasts fresh from academics who wish to embark upon a career in analytical industry. Hi Ramesh, During the derivatization process it appears that some impurities are getting introduced.
As unidentified impurities are the main contributors to noise try using derivatization reagent of higher purity. Also take care to degas the mobile phase thoroughly as minute air bubbles can also give rise to random noise. My mail id deepak. Magnificent goods from you, man. You make it entertaining and you still take care of to keep it sensible. I cant wait to read far more from you.
This is really a tremendous site. Do you have any recommendations for newbie blog writers?Achieving a flat baseline which does not exhibit spikes, ghost peaks, drift or wander in an unpredictable manner should be a primary goal when performing HPLC analysis or developing methods.
Methods which result in flat baselines and have well defined, sharp peaks allow for accurate sample area integration. Integration algorithms perform poorly in quantifying peaks on sloped, drifting or noisy baselines. Properly developed HPLC methods are reproducible methods which apply and utilize good chromatography fundamentals.
Note: A lack of proper training in the operation of the HPLC system, improper start-up or poor quality maintenance of the chromatograph Examples: failure to degas and purge the system lines before use; an air bubble stuck in a check valve, a bad detector lamp or a leak will often result in baseline noise are the main causes of noise. Your HPLC system must be optimized for your specific application. Be sure and allow time for the mobile phase to reach full equilibration with the system before starting any analysis.
In this article, we will discuss how temperature fluctuations, inadequate mixing, inadequate degassing and flow cell contamination can result in excessive baseline noise. We will provide suggestions on how to reduce or eliminate these problems.
To reduce temperature fluctuations, you must control the temperature of the column and mobile phase if applicable during the analysis. This is most commonly done by: a using equilibrated mobile phase at the start of the day or analysis, b keeping the interconnecting lines as short as possible esp.
Control of the column temperature will remove 'temperature' as a variable from your analysis. Temperature should be a constant run to run, not a variable. Be sure and document the temperature selected as part of your method.
The associated noise and ripple of incomplete mixing can reduce the limit of detection LOD and increase integration error. Both high pressure with separate pumps and low pressure pumping one pump with a multi-channel proportioning valve systems depend on efficient mixing to reduce noise. For gradient analysis, failure to completely mix the mobile phase solution before it enters the HPLC column often results in excessive baseline noise, spikes and poor reproducibility.
Mixing also performed directly in a mixer installed in the flow path of an HPLC pump. This mixer is often a static mixer a simple 'Tee', a tube filled with baffles, a frit or beads, valve orifice or microfluidic device of low volume design for chromatography use, but allows adequate mixing of the liquids within a prescribed flow rate range.
The best mixers incorporate longitudinal and radial mixing in-line. A mixer with too low a volume or of insufficient design can result in poor mixing of the mobile phase note: incorrect solvent compressibility settings can also cause mixing and noise problems too. To reduce mixing problems, first insure that the mobile phases used are fully soluble with each other. Next, make sure that any mixer used is appropriate for the flow rates and volumes you will be using. If needed, run a gradient valve test to insure that each valve channel is working properly, not leaking or introducing any cross-flow leakage to another channel.
For the best results, continuously degas your mobile phase. Reducing the amount of gas will also improve signal to noise levels of detection, reduce drift and reduce pump cavitation. A faulty degasser may cause more damage contamination to your system and methods.
Maintain and Repair them just as you do for your other instrument modules. To achieve the best balance of low noise levels and high reliability, both aqueous and organic mobile phases should be fully degassed before and during use.
Never use Nitrogen or Argon gasthey are soluble in the liquid! In all cases, degassing must be continuous not just done one time. Continuous degassing reduces cyclical noise and signal variations. For this reason, I do not recommend using ultrasonic baths to degass mobile phase solutions as these are not used in a continuous mode.Resolution of chromatography
The mobile phase solution starts to re-absorb gas as soon as you stop sonicating the solution. This results in continuous baseline drift up and down. Removal of gasses is critical to the function of a modern HPLC pumping system. The liquids used are compressed to very high levels which forces out solubilized gas from the solutions. This is best accomplished before the liquid is transferred into the pump.You should find out, what caused high back pressure - column or system?
We recommend following procedure:. Remove column from the system and turn on pump. If high back pressure still appears, then the blockage is in the system:. Leaks are usually stopped by tightening or replacing a fitting. Be aware, however, that overtightened metal compression fittings can leak and plasrtic fingertight fittings can wear out. If a fitting leak does not stop when the fitting is tightened a little, take the fitting apart and inspect for damage e.
If the fitting or ferrule is damaged, replace it with new one. Many issues in the LC system appear as chenges in the chromatogram. Some of these can be solved by changes in the instrument, however, other problems require modification of the assay procedure. Setting the proper column type, pre-column or guard column, tubings, detector cell and mobile phase are keys to good chromatography.
The distortion of early eluting peaks can be caused by wrong injection solvent. Reduce the injection volume, or use weaker injection solvent. Broad peaks can be caused by extra-column effects:. Company certificates Trade conditions News. Chromatography Gas detection Physical property measurement.
Contact data E-mail contact form. News Chromservis. Backflush column if it is permitted, replace frit according to the manufacturer's instructions and warratny conditions or replace column. Loosen and retighten the fitting. If the fitting is damaged, replace it. Disassemble fitting and clean it.
Reverse flush column if it is allowed or replace frit if it is allowed or replace column. Adjust pH. For basic compounds, lower pH usually provides more symmetric peaks. Reverse flush column if it is allowed or replace inlet frit if it is aalowed or replace the column. Remove guard column and attempt analysis. If analytical column is obstructed, reverse and flush if it is allowed.
If problem persists, column may be fouled with strongly retained contaminants. Use appropriate restoration procedure see column care information. If problem persists, inlet is probably plugged. Change frit or replace column. Replumb the system shorter, narrower tubinguse smaller volume detector cell. Add triethylamine basic sampes or add acetate acidic samples or add salt or buffer ionic samples or try a different column.
Add triethylamine basic compounds or add acetic acid acidic compounds or add water poly-functional compounds.
Only for normal-phase methods which use water-miscible solvents. You can also try a different LC method. Use 50 to mM buffer concentration, use buffer with pKa equal to pH of mobile phase.
Impurities in the sample, reagents or material used. Column temperature fluctuation.Many 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.
Problems with the Chromatogram Many problems in an LC system show up as changes in the chromatogram. Peak Tailing Possible Cause Solution. Replace inlet frit. Replace column. For basic compounds, a lower pH. Change column. See A. Replace guard if necessary c. If analytical column is obstructed, reverse and flush d. If problem persists, column may be fouled with strongly retained contaminants e.
Use appropriate restoration procedure f. If problem persists, inlet is probably plugged g. Change frit or replace column. Use weaker injection solvent. Use smaller volume detector cell. Add acetate acidic samples.
Add salt or buffer ionic samples. Try a different column. Add acetic acid. Use 50— m m buffer concentration. Use buffer with pKa equal to pH of. Increase flow rate. Use mobile phase as injection solvent. Reduce injection volume. Set proper mobile phase mixture on.
Use heat exchanger before detector. UV detectors at high sensitivity. Degas mobile phase before use. Sparge with helium during use.Chromatograms are like fingerprints. In this series, we will show a series of GC-chromatograms that are obtained from users and discuss some potential causes for the phenomena.
Then we can move into some solutions for improvement. When using GC sometimes the baseline increases with every analysis. Built up of later eluting peaks in the column.
If there are heavy components in the sample, they will elute eventually. You can follow any responses to this entry through the RSS 2. You can leave a responseor trackback from your own site. Name required.
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