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TROUBLESHOOTING IN HIGH PERFORMANCE LIQUID CHROMATOGRAPHY


About Authors:
Ipshita Chattopadhyaya, Ekta Malhotra
Department of Pharmacy,
Jaipur National University,
Jaipur, India

ABSTRACT
(1)The developments in column packing technology and suitable equipment paved the way for what is now called High Performance or High Pressure Liquid Chromatography (HPLC). The new technique provided much higher resolution, more accurate quantitative results, as well as shorter analysis times in comparison to the earlier techniques .During the sixties, new theoretical insights accompanied by important. Since its introduction, HPLC has evolved into an indispensable tool in many analytical laboratories and is applied to diverse analytical problems. Actually, HPLC refers to a number of separation techniques that use a liquid mobile phase, or eluent. Troubleshooting HPLC instrumentation and separations require a fundamental understanding of how the instrument functions and how the separation works. (2)This article provides a practical guide to common HPLC problems, along with more in-depth information to help the reader understand the relationships between the observed symptoms and the underlying causes. The practical approach presented here is meant to serve as both a troubleshooting guide and an HPLC learning tool.

Reference Id: PHARMATUTOR-ART-1190

ABOUT HPLC
(3)
Prior to the 1970's, few reliable chromatographic methods were commercially available to the laboratory scientist. During 1970's, most chemical separations were carried out using a variety of techniques including open-column chromatography, paper chromatography, and thin-layer chromatography. However, these chromatographic techniques were inadequate for quantification of compounds and resolution between similar compounds. (4)During this time, pressure liquid chromatography began to be used to decrease flow through time, thus reducing purification times of compounds being isolated by column chromatography. However, flow rates were inconsistent, and the question of whether it was better to have constant flow rate or constant pressure was debated. (5)High pressure liquid chromatography was developed in the mid-1970's and quickly improved with the development of column packing materials and the additional convenience of on-line detectors. In the late 1970's, new methods including reverse phase liquid chromatography allowed for improved separation between very similar compounds (6)By the 1980's HPLC was commonly used for the separation of chemical compounds. New techniques improved separation, identification, purification and quantification far above the previous techniques. Computers and automation added to the convenience of HPLC. Improvements in type of columns and thus reproducibility were made as such terms as micro-column, affinity columns, and Fast HPLC began to emerge. (7)The past decade has seen a vast undertaking in the development of the micro-columns, and other specialized columns. (8)The dimensions of the typical HPLC column are: 250 mm in length with an internal diameter between 3-5 mm. hope that this article will provide readers with HPLC HISTORY its operation parameters and handle trouble while operation.

TROUBLES FACED DURING HPLC OPERATIONS AND ITS SOLUTIONS

Problem

Possible cause

Solution

(10)No peaks or very small peaks

Detector off

Check detector

Broken connections to recorder

Check connections

No sample/Wrong sample

Check sample. Be sure it is not deteriorated. Check for bubbles in the vials

Wrong settings on recorder or detector

Check attenuation. Check gain

No Flow

Pump off

Start Pump

Flow interrupted

Check reservoirs. Check position of the inlet tubing. Check loop for obstruction or air. Check degasing of mobile phase. Check compatibility of the mobile phase components.

Leak

Check fittings. Check pump for leaks and precipitates. Check pump seals.

Air trapped in the system

Disconnect column and prime pump. Flush system with 100% methanol or isopropanol. Contact servicing if necessary.


Column and Fittings Leaks

Problem

Possible cause

Solution

Column end leaks

Loose fitting 
White powder at loose fitting

Tighten or replace fitting 
Cut tubing and replace ferrule; disassemble fitting, rinse and reassemble.

Leak at detector

Detector-seal failure

Replace detector seal or gaskets.

Leak at injection valve

Worn or scratched valve rotor

Replace valve rotor

Leak at pump

Pump seal failure

Replace pump seal; check piston for scratches and, if necessary, replace

Change in Retention time

Problem

Possible cause

Solution

Changing Retention Times

Buffer retention times

Use buffer with concentration greater than 20 mM.

Contamination buildup

Flush column occasionally with strong solvent

Equilibration time insufficient for gradient run or changes in isocratic mobile phase

Pass at least 10 column volumes through the column for gradient regeneration or after solvent changes

First few injections - active sites

Condition column by injecting concentrated sample

Inconsistent on-line mobile-phase mixing

Ensure gradient system is delivering a constant composition; compare with manually prepared mobile phase; partially premix mobile phase

Selective evaporation of mobile-phase component

Cover solvent reservoirs; use less-vigorous helium purging; prepare fresh mobile phase

Varying column temperature

Thermostat or insulate column; ensure laboratory temperature is constant.

Decreasing Retention Times

Active sites on column packing

Use mobil-phase modifier, competing base (basic compounds), or increase buffer strength; use higher coverage column packing.

Column overloaded with sample

Decrease sample amount or use larger-diameter column.

Increasing flow rate

Check and reset pump flow rate.

Loss of bonded stationary phase or base silica

Use mobile-phase pH between pH 2 and pH 8

Varying column temperature

Thermostat or insulate column; ensure laboratory temperature is constant

Increasing Retention Times

Decreasing flow rate

Check and reset pump flow rate; check for pump cavitation; check for leaking pump seals and other leaks in system

Changing mobile-phase composition

Cover solvent reservoirs; ensure that gradient system is delivering correct composition.

Loss of bonded stationary phase

Use mobile-phase pH between pH 2 and pH 8

Slow column equilibration time

Reversed phase ion pairing - long chain ion pairing reagents require longer equilibration time

Use ion-pairing reagent with shorter alkyl chain length

 

                                                           Baseline

Problem

Possible cause

Solution

Void Time noise

Air bubbles in mobile phase

Degas or use back pressure restricor on detector

Positive-negative - difference in refractive index of injection solvent and mobile phase

Normal with many samples; use mobile phase as sample solvent

Drifting baseline

Negative direction (gradient elution) - absorbance of mobile-phase A

Use non-UV absorbing mobile phase solvents; use HPLC grade mobile phase solvents; add UV absorbing compound to mobile phase B.

Positive direction (gradient elution) - absorbance of mobile phase B

Use higher UV absorbance detector wavelength; use non-UV absorbing mobile phase solvents; use HPLC grade mobile phase solvents; add UV absorbing compound to modile phase A.

Positive direction - contamination buildup and elution

Flush column with strong solvent; clean up sample; use HPLC grade solvents

Wavy or undulating - temperature changes in room

Monitor and control changes in room temperature; insulate column or use column oven; cover refractive index detector and keep it out of air currents.

(11)Baseline noise

Continous - detector lamp problem or dirty cell

Replace UV lamp( each should last 2000 h; clean and flush flow cell.

Gradient or isocratic proportioning - lack of solvent mixing

Use proper mixing device; check proportioning precision by spiking one solvent with UV absorbing compound and mointor UV absorbance detector outputl.

Gradient or isocratic proportioning - malfunctioning proportioning valvesl

Clean or replace proportioning precision valves; partially remix solventsl.

Occasional sharp spikes - external electrical interference

Use voltage stabilizer for LC system; use independent electrical circuit.

Periodic - pump pulses

Service or replace pulse damper; purge air from pump; clean or replace check valves.

Random - contamination buildup

Flush column with strong solvent; clean up sample; use HPLC grade solvent

Spikes - bubble in detector

Degas mobile phase; use back pressure restrictor at detector outlet.

Spikes - column temperature higher than boiling point of solvent

Use lower column temperature.

 

Pressure

Problem

Possible cause

Solution

Decreasing Pressure

Insufficient flow from pump

Loosen cap on mobile phase reservior

Leak in hydralic lines from pump to column

Tighten or replace fittings; tighten rotor in injection valve

Leaking pump check valve or seals

Replace or clean check valves; replace pump seals.

Pump cavitation

Degas solvent; check for obstruction in line from solvent reservoir to pump; replace inlet-line frit

Fluctuating pressurre

Bubble in pump

Degas solvent; purge solvent with helium

Leaking pump check valve or seals

Replace or clean check valves; replace pump seals

High Back Pressure

Column blocked wth irreversibly adorbed sample

Improve sample cleanup; use guard column; reverse-flush column with strong solvent to dissolve blockage

Column particle size too small (for example 3 micrometers)

Use larger particle size (for example 5 micrometer)

Microbial growth on column

Use at least 10% organic modifier in mobile phase; use fresh buffer daily; add 0.02% sodium azide to aqueous mobile phase; store column in at least 25% organic solvent without buffer

Mobile phase viscosity too high

Use lower viscosity solvents or higher temperature

Plugged frit in in-line filter or guard column

Replace frit or guard column

Plugged inlet frit

Replace endfitting or frit assembly

Polymetric columns - solvent change causes swelling of packing

Use correct solvent with column; change to proper solvent compositionl consult manufacturer's solvent-compatibility chartl use a column with a higher percentage of cross-linking

Salt precipitation (especially in reversed-phase chromatography with high concentration of organic solvent in mobile phase) concentration of organic solvent in mobile phase)

Ensure mobile phase compatibility with buffer concentration; decrease ionic strength and water-organic solvent ratio; premix mobile phase

When injector disconnected from column - blockage in injector

Clean injector or replace rotor

Increasing Pressure

Blocked flow lines

Systematically disconnect components from detector end to column end to find blockage; replace or clean blocked component

Particulate buildup at head of column

Filter sample; use .5 micrometer in-line filter; disconnect and backflush column; replace inlet frit

Water-organic solvent systems - buffer precipitation

Ensure mobile phase compatibility with buffer concentration; decrease ionic strength or water organic solvent ratio

 

Peaks

Problem

Possible cause

Solution

Broad peaks

Analytes eluted early due to sample overload

Dilute sample 1:10 and reinject

Detector-cell volume too large

Use smallest possible cell volume consistent with sensitivity needs; use detector with no heat exchanger in system

Injection volume too large

Decrease solvent strength of injection solvent to focus solute; inject smaller volume

Large extra column volume

Use low- or zero-dead-volume endfittings and connectors; use smallest possible diameter of connecting tubing (<0.10 in. i.d.); connect tubing with matched fittings

Mobile-phase solvent viscosity too high

Increase column temperature; change to lower viscosity solvent

Peak dispersion in injector valve

Decrease injector sample loop size; introduce air bubble in front and back of sample in loop

 

Poor column efficiency

Use smaller-particle-diameter packing, lower-viscosity mobile phase, higher column temperature, or lower flow rate

 

Retention time too long

Use gradient elution or stronger isocratic mobile phase

 

Sampling rate of data system too low

Increase sampling frequency.

 

Slow detector time constant

Adjust time constant to match peak width

 

Some peaks broad - late elution of analytes retained from previous injection

Flush column with strong solvent at end of run; end gradient at higher solvent concentration

 

Ghost peaks

Contamination

Flush column to remove contaminatint; use HPLC-grade solven

 

Elution of analytes retained from previous injection

Flush column with strong solvent at end of run; end gradient at higher solvent concentration

 

Ion-pair chromatography - upset equilibrium

Prepare sample in mobile phase; reduce injection volume

 

Oxidation of trifluoroacetic acid in peptide mapping

Prepare trifluoroacetic acid solutions fresh daily; use antioxidant

 

Reversed-phase chromatography - contaminated water

Check suitability of water by running different amounts through column and measure peak height of interferences as function of enrichment time; clean water by running it through old reversed-phase column; use HPLC-grade water.

 

Unknown interferences in sample

Use sample cleanup or prefractionation before injection.

 

Negative peaks

Refractive index detection - refractive index of solute less than that of mobile phase

Reverse polarity to make peak positive

 

UV-absorbance detection - absorbance of solute less than that of mobile phase

Use mobile phase with lower UV absorbance; if recycling solvent, stop recycling when recycled solvent affects detection

 

Peak Doubling

Blocked Frit

Replace or clean frit; install 0.5-um porosity in-line filter between pump and injector to eliminate mobile-phase contaminants or between injector and column to eliminate sample contaminants

 

Coelution of interfering compound

Use sample cleanup or prefractionation; adjust selectivity by changing mobile or stationary phase

 

Coelution of interfering compound from previous injection

Flush column with strong solvent at end of ran; end gradient at higher solvent concentration

 

Column overloaded

Use higher-capacity stationary phase; increase column diameter; decrease sample amount

 

Column void or channeling

Replace column, or, if possible, open top endfitting and clean and fill void with glass beads or same column packing; repack column

 

Injection solvent too strong

Use weaker injection solvent or stronger mobile phase

 

Sample volume too large

Use injection volume equal to one-sixth of column volume when sample prepared in mobile phase for injection

 

Unswept injector flow path

Replace injector rotor

 

Peak Fronting

Channeling in column

Replace or repack column

 

Column overloaded

Use higher-capacity stationary phase; increase column diameter; decrease sample amount

 

Tailing Peaks

Basic solutes - silanol interactions

Use competing base such as triethylamine; use a stronger mobile phase; use base-deactivated silica-based reversed-phase column; use polymeric column

 

Beginning of peak doubling

See peak doubling

 

Chelating solutes - trace metals in base silica

Use high purity silica-based column with low trace-metal content; add EDTA or chelating compound to mobile phase; use polymeric column

 

Silica-based column - degradation at high pH

Use polymeric, sterically protected, or high-coverage reversed-phase column; install silica gel saturatorcolumn between pump and injector

 

Silica-based column - degradation at high temperature

Reduce temperature to less than 50 C

 

Silica-based column - silanol interactions

Decrease mobile-phase pH to suppress silanol ionization; increase buffer concentration; derivatize solute to change polar interactions

 

Unswept dead volume

Minimize number of connections; ensure injector rotor seal is tight; ensure all compression fittings arecorrectly seated

 

Void formation at head of column

Replace column, or, if possible, open top endfitting and clean and fill in void with glass beads or samecolumn packing; rotate injection valve quickly; use injection valve with pressure bypass; avoid pressure shock

 

Spikes

Bubbles in mobile phase

Degas mobile phase; use back-pressure restrictor at detector outlet; ensure that all fittings are tight

 

Column stored without caps

Store column tightly capped; flush reversed-phase columns with degassed methanol

 

CONCLUSION
GC and HPLC both have a place in research, product assessment, and environmental monitoring. While GC works best with analytes below 1000 daltons, HPLC is suited to separating higher molecular weight compounds in order to provide qualitative and quantitative information. HPLC chromatographic separations and separatory systems are characterized by the retention and resolution of analyte peaks, as well as their selectivity and plate number (efficiency). Hence the precision and accuracy needed for the assessment of the substances throh HPLC solely depends on the instrument and the ability to rectify the instrumentalproblems.

REFERENCE
INTERNET
1.waters.com visited on 11 november 2011 at 3:00 pm Kolkata.
2. sigmaldrich.com visited on 11 november 2011 at 3:35pm Kolkata  
3.  google.books.co.in visited on 12 november 2011 at 11:00 am at Kolkata.
4.  forumsci.com visited on 12 november 2011 at 11: am at Kolkata.
BOOKS
5. Miachel w.Dong. Modern HPLC for  practicing Scientist. Page no.275.10 second edition.
6. Stravos Kromidas ,HPLC Made to Measure ,page no.753, first edition.
7. Satinder Ahuja ,A Handbook of  Pharmaceutical Analysis, page no.255, third edition.
8. Gunter J.Eppert,HPLC Trouble shooting,pageno.98 third edition.
9. Lylod .R.Sinder, John W Dolan, High Performance Gradient Elution Practical Approach to HPLC page no.153 third edition.
10. Dean Rood ,A Practical Guide to the Care and Maintenance of  HPLC.first edition.
11. Journal of High Resolution Chromatography VOL 20.Page 120.second edition.

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