a guide to problem-solving in high performance liquid...

Journal of Pharmacy Research Vol.5 Issue 8.August 2012

Pankti P. Desai et al. / Journal of Pharmacy Research 2012,5(8),4411-4420

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Review ArticleISSN: 0974-6943

Available online throughhttp://jprsolutions.info

*Corresponding author.Pankti DesaiDepartment of Pharmaceutical Analysis,Ramanbhai Patel College of Pharmacy,Charotar University of Science andTechnology, CHARUSAT Campus,Changa-388421, Gujarat, India

INTRODUCTIONThe article contains information on the most likely cause(s) of the observedproblem and ways in which they can be rectified. Basic troubleshootinginvolves systematically isolating the problem. Once the problem is isolated,the next step is to change only one variable at a time to see if it resolves theproblem. After each change, the whole system should be checked again todetermine whether the problem still exists or whether the change correctedthe problem. All problems and corrective action should be documented inthe instrument log, for future reference.

Troubleshooting strategy involves five steps: (1) Identification of theproblem; (2) Awareness of the cause of the problem; (3) Isolation of theexact cause of the problem; (4) Rectifying the problem; and (5) Returningthe unit to routine use.

Commonly encountered problems and their solutionsA systematic approach is best to identify any problems whentroubleshooting the high-performance liquid chromatography (HPLC)system. This article is organized into five major categories of problems tohelp quickly identify the source of the problem(s): (1) Poor Peak Shape; (2)Pressure Problems; (3) Baseline Problems; (4) Variable Retention time; and(5) Miscellaneous Problems.

Poor Peak ShapeAn ideal chromatogram has Gaussian peaks. The peak shape is importantparameter as it is a reflection of the processes occurring within thechromatographic system. Thus, it acts as an indicator of chromatographicproblems (instrumental or method related). The most common indicationsare shown in figure 1. The main causes of each problem along with itsremedy are described in the table I.

Pressure ProblemsSystem pressure is affected by a number of variables including the viscosity

A Guide to Problem-Solving in High Performance Liquid ChromatographyPankti P. Desai*1, Kunti, D. Raskapur2

1Department of Pharmaceutical Analysis, Ramanbhai Patel College of Pharmacy, Charotar University of Science and Technology, CHARUSAT Campus,Changa-388421, Gujarat, India

2Department of Quality Assurance, Ramanbhai Patel College of Pharmacy, Charotar University of Science and Technology, CHARUSAT Campus, Changa-388421, Gujarat, India

Received on:11-05-2012; Revised on: 16-06-2012; Accepted on:19-07-2012

ABSTRACTThe article is intended to aid in troubleshooting problems that occur more often with the High Performance Liquid Chromatography (HPLC) systemduring routine analysis. Primary aim is to focus the commonly encountered problems along with more in-depth information to help the reader understandthe relationships between the observed symptoms, the underlying causes, and their corrective actions, to maximize system operation time and to promotegood chromatographic practices.

Key words: HPLC, poor peak shape, baseline problems, pressure problems, variable retention time.

of the solvent used, column variables, flow rate and temperature. It isimportant to have a reference point when high or low pressures to the norm.This reference point should be the pressure generated in the system wheneverything if functioning correctly. Pressure problems fall into one of threecategories: high, low or fluctuating pressure. They can occur suddenly or bea gradual process. Sudden pressure rises tend to be due to particles from thesample, blocked or damaged tubing or column packed bed collapse. Gradualpressure rises can also be due to particles in the sample, but they can alsoarise from particles generated in the instrument, for example, debris fromvial septa or degrading seals. The simplest way to troubleshoot pressureproblems is using a systematic approach, as highlighted in table II for high,low or fluctuating pressure.

A] Low PressureThe most common causes of no/low pressure are the solvent inlet lines notbeing immersed in solvent, no solvent in the reservoir and leaks.

B] Fluctuating pressureThe most common cause of fluctuating pressure is poorly primed lines withbadly degassed solvents.

C] High PressureThe most common causes of high pressure are blocked tubing around theinjector and column inlet.

Baseline problemsBaseline irregularities can be non-cyclic or cyclic. They can originate fromelectrical interferences, detector faults, solvent impurities, columncontamination, etc. To isolate the source of a baseline irregularity, it isimportant to determine whether the problem lies with the fluid path, detectoror electrical connections. This can be achieved by following the simplesteps: (1) Turn off the instrument pump - fluid flow must be zero. (2)Monitor the baseline for 5 to 10 min. note if there is any improvement in thebaseline’s appearance. If yes, then the problem lies within the instrumentfluid path. If no, the problem is either electrical or detector related. (3)Disconnect the detector electrical cables from the A/D interface with PC,integrator and chart recorder, i.e. the data handling devices. Attach a jumpsource to the input terminals on the data-handling device (a crocodile clip,paper clip). If the noise continues, then the problem is within the data



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Table I: Possible causes and remedy for poor peak shape



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Table II: Possible causes and remedy for pressure problems



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Table III: Possible causes and remedy for baseline problems



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Table IV: Possible causes and remedy for variable retention time



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Table V: Miscellaneous problems

Figure 1: Peak shape indications



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Figure 2: Baseline Problems Figure

Figure 3: Variable Retention Time Figure

handling device. The commonly encountered baseline problems are shownin Figure 3 and table III.

Variable Retention TimeThe most common cause of peak retention time drift in one direction ispoorly prepared or mixed solvents or a system leak. If the solvents wereprepared correctly, then it is very important to determine whether they arebeing mixed correctly (mixing cell problems). Where solvents are mixedmanually prior to pumping, ensure that the solvent flow rate is correct andconstant. Figure 3 represents effects of variable retention time. (Table IV)

Miscellaneous ProblemsThe other types of problems that are likely to occur are described table V.

CONCLUSIONIt has been rightly quoted that prevention is better than cure. This articlesummarizes the most routinely encountered problems when working withHPLC. The problems are evident as high pressure, undesirable peak shapeand changes in retention/selectivity, baseline problems. Often these problemsare not associated with the column and may be caused by instrument andchemistry issues, pH of mobile phase, instrument connections, detectorsettings and metal contamination. It is recommended to start with thecorrect questions, find the answers, and the answers will surely lead tosolutions.

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Source of support: Nil, Conflict of interest: None Declared

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a guide to problem-solving in high performance liquid...

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