Introduction

High-performance liquid chromatography

(Or High pressure liquid chromatography, HPLC) HPLC is a popular method of analysis because it is easy to learn and use and is not limited by the volatility or stability of the sample compound. An analytical technique used for separation of low-to-moderate molecular weight compounds of resins. The instrumentation for HPLC and size exclusion (SEC) or gel-permeation chromatography are similar, but the columns differ. It is a form of column chromatography used frequently in biochemistry and analytical chemistry to separate, identify, and quantify compounds. HPLC utilizes a column that holds chromatographic packing material (stationary phase), a pump that moves the mobile phase(s) through the column, and a detector that shows the retention times of the molecules. Retention time varies depending on the interactions between the stationary phase, the molecules being analyzed, and the solvent(s) used.

Fig. 1 High-performance liquid chromatography

HISTORY

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 opencolumn chromatography, paper chromatography, and thinlayer chromatography. However, these chromatographic techniques were inadequate for quantification of compounds and resolution between similar compounds. During this time, pressure liquid chromatography began to be used to decrease flowthrough time, thus reducing purification times of compounds being isolated by column chromatogaphy. However, flow rates were inconsistant, and the question of whether it was better to have constant flow rate or constant pressure was debated. 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. 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 immerge. The past decade has seen a vast undertaking in the development of the micro-columns, and other specialized columns. The dimensions of the typical HPLC column are: XXX mm in length with an internal diameter between 3-5 mm. The usual diameter of micro-columns, or capillary columns, ranges from 3 m to 200 m. Fast HPLC utilizes a column that is shorter than the typical column, with a length of about 3 mm long, and they are packed with smaller particles. Currently, one has the option of considering over x# types of columns for the separation of compounds, as well as a variety of detectors to interface with the HPLC in order to get optimal analysis of the compound. We hope this review will provide a reference which all levels of HPLC users will be able to find quick answers to their HPLC problems. Although HPLC is widely considered to be a technique mainly for biotechnological, biomedical, and biochemical research as well as for the pharmaceutical industry, these fields corrently comprise only about 50% of HPLC users. Liquid chromatography was defined in the early 1900s by the work of the Russian botanist, Mikhail S. Tswett. His pioneering studies focused on separating compounds [leaf pigments], extracted from plants using a solvent, in a column packed with particles.

Tswett filled an open glass column with particles. Two specific materials that he found useful were powdered chalk [calcium carbonate] and alumina. He poured his sample [solvent extract of homogenized plant leaves] into the column and allowed it to pass into the particle bed. This was followed by pure solvent. As the sample passed down through the column by gravity, different colored bands could be seen separating because some components were moving faster than others. He related these separated, different-colored bands to the different compounds that were originally contained in the sample. He had created an analytical separation of these compounds based on the differing strength of each compounds chemical attraction to the particles. The compounds that were more strongly attracted to the particles slowed down, while other compounds more strongly attracted to the solvent moved faster. This process can be described as follows: the compounds contained in the sample distribute, or partition differently between the moving solvent, called the mobile phase, and the particles, called the stationary phase. This causes each compound to move at a different speed, thus creating a separation of the compounds. Tswett coined the name chromatography [from the Greek words chroma, meaning color, and graph, meaning writingliterally, color writing] to describe his colorful experiment. [Curiously, the Russian name Tswett means color.] Today, liquid chromatography, in its various forms, has become one of the most powerful tools in analytical chemistry.

Figure A: Tswett's Experiment

Liquid Chromatography [LC] Techniques

Liquid chromatography can be performed using planar [Techniques 1 and 2] or column techniques [Technique 3]. Column liquid chromatography is the most powerful and has the highest capacity for sample. In all cases, the sample first must be dissolved in a liquid that is then transported either onto, or into, the chromatographic device. Technique 1. The sample is spotted onto, and then flows through, a thin layer of chromatographic particles [stationary phase] fixed onto the surface of a glass plate [Figure B]. The bottom edge of the plate is placed in a solvent. Flow is created by capillary action as the solvent [mobile phase] diffuses into the dry particle layer and moves up the glass plate. This technique is called thin-layer chromatography or TLC.

Figure B: Thin-layer Chromatography

APPLICATION FOR HPLC

Preparative HPLC refers to the process of isolation and purification of compounds. Important is the degree of solute purity and the throughput, which is the amount of compound produced per unit time. This differs from analytical HPLC, where the focus is to obtain information about the sample compound. The information that can be obtained includes identification, quantification, and resolution of a compound. Chemical Separations can be accomplished using HPLC by utilizing the fact that certain compounds have different migration rates given a particular column and mobile phase. Thus, the chromatographer can separate compounds (more on chiral separations) from each other using HPLC; the extent or degree of separation is mostly determined by the choice of stationary phase and mobile phase. Purification refers to the process of separating or extracting the target compound from other (possibly structurally related) compounds or contaminants. Each compound should have a characteristic peak under certain chromatographic conditions. Depending on what needs to be separated and how closely related the samples are, the chromatographer may choose the conditions, such as the proper mobile phase, to allow adequate separation in order to collect or extract the desired compound as it elutes from the stationary phase. The migration of the compounds and contaminants through the column need to differ enough so that the pure desired compound can be collected or extracted without incurring any other undesired compound.

--HPLC of Proteins and Polynucleotides

Identification of compounds by HPLC is a crucial part of any HPLC assay. In order to identify any compound by HPLC a detector must first be selected. Once the detector is selected and is set to optimal detection settings, a separation assay must be developed. The parameters of this assay should be such that a clean peak of the known sample is observed from the chromatograph. The identifying peak should have a reasonable retention time and should be well separated from extraneous peaks at the detection levels which the assay will be performed. To alter the retention time of a compound, several parameters can be manipulated. The first is the choice of column, another is the choice of mobile phase, and last is the choice in flow rate. All of these topics are reviewed in detail in this document. Identifying a compound by HPLC is accomplished by researching the literature and by trial and error. A sample of a known compound must be utilized in order to assure identification of the unknown compound. Identification of compounds can be assured by combining two or more detection methods. Quantification of compounds by HPLC is the process of determining the unknown concentration of a compound in a known solution. It involves injecting a series of known concentrations of the standard compound solution onto the HPLC for detection. The chromatograph of these known concentrations will give a series of peaks that correlate to the concentration of the compound injected.

USES
HPLC stands for high performance liquid chromatography. It is a liquid chromatography which involves the separation of the compounds on the basis of their polarity. It is used to analyze, identify, purify & quantify the compounds. HPLC is one of the most widely applied analytical separation techniques.It is used for chemistry and biochemistry research analyzing complex mixtures, purifying chemical compounds, developing processes for synthesizing chemical compounds, isolating natural products, or predicting physical properties. It is also used in quality control to ensure the purity of raw materials, to control and improve process yields, to quantify assays of final products, or to evaluate product stability and monitor degradation. In addition, it is used for analyzing air and water pollutants, for monitoring materials that may jeopardize occupational safety or health, and for monitoring pesticide levels in the environment. Federal and state regulatory agencies use HPLC to survey food and drug products, for identifying confiscated narcotics or to check for adherence to label claims. Pharmaceutical Tablet dissolution of pharmaceutical dosages Shelf-life determinations of pharmaceutical products Identification of counterfeit drug products Pharmaceutical quality control

Environmental Phenols in Drinking Water Identification of diphenhydramine in sediment samples Biomonitoring of PAH pollution in high-altitude mountain lakes through the analysis of fish bile Estrogens in coastal waters - The sewage source Toxicity of tetracyclines and tetracycline degradation products to environmentally relevant bacteria Assessment of TNT toxicity in sediment

Forensics A mobile HPLC apparatus at dance parties - on-site identification and quantification of the drug Ecstasy Identification of anabolic steroids in serum, urine, sweat, and hair Forensic analysis of textile dyes Determination of cocaine and metabolites in meconium Simultaneous quantification of psychotherapeutic drugs in human plasma

Clinical Quantification of DEET in Human Urine Analysis of antibiotics Increased urinary excretion of aquaporin 2 in patients with liver cirrhosis Detection of endogenous neuropeptides in brain extracellular fluids

Food and Flavor

Ensuring soft drink consistency and quality Analysis of vicinal diketones in beer Sugar analysis in fruit juices Polycyclic aromatic hydrocarbons in Brazilian vegetables and fruits Trace analysis of military high explosives in agricultural crops Stability of aspartame in the presence of glucose and vanillinconcentration is found by solving the equation for x.

OPERATION

The sample to be analyzed is introduced in small volume to the stream of mobile phase. The analyte's motion through the column is slowed by specific chemical or physical interactions with the stationary phase as it traverses (passes through) the length of the column. The amount of retardation depends on the nature of the analyte, stationary phase and mobile phase composition. The time at which a specific analyte elutes (comes out of the end of the column) is called the retention time; the retention time under particular conditions is considered a reasonably unique identifying characteristic of a given analyte. The use of smaller particle size column packing (which creates higher backpressure) increases the linear velocity (speed) giving the components less time to diffuse within the column, leading to improved resolution in the resulting chromatogram. Common solvents used include any miscible combination of water or various organic liquids (the most common are methanol and acetonitrile). Water may contain buffers or salts to assist in the separation of the analyte components, or compounds such as trifluoroacetic acid which acts as an ion pairing agent.

A further refinement to HPLC has been to vary the mobile phase composition during the analysis; this is known as gradient elution. A normal gradient for reversed phase chromatography might start at 5% methanol and progress linearly to 50% methanol over 25 minutes; the gradient chosen depends on how hydrophobic the analyte is. The gradient separates the analyte mixtures as a function of the affinity of the analyte for the current mobile phase composition relative to the stationary phase. This partitioning process is similar to that which occurs during a liquidliquid extraction but is continuous, not step-wise. In this example, using a water/methanol gradient, the more hydrophobic components will elute (come off the column) when the mobile phase consists mostly of methanol (giving a relatively hydrophobic mobile phase). The more hydrophilic compounds will elute under conditions of relatively low methanol/high water. The choice of solvents, additives and gradient depend on the nature of the stationary phase and the analyte. Often a series of tests are performed on the analyte and a number of trial runs may be processed in order to find the HPLC method which gives the best separation of peaks.

CALIBRATION
HPLC calibration the expressions lowest calibration limit and determination limit are defined in statistical terms. The lowest calibration limit is the minimum mass in the measured series of calibration points. It is calculated from the confidence interval of the inverse of the calibration function as the lowest mass limit that may be differentiated from zero mass with a preset probability of error. If the calculated lowest calibration limit is lower than the actual data, points at lower concentration may be measured. The determination limit is the smallest concentration of an analysis that is differentiated from the concentration zero or an apparent blind value in the calibration curve with a given probability of error.Using two different UV-detectors (variable wavelength and photodiode-array) the lowest calibration limit is experimentally evaluated and compared with specific data for the detectors. Chromatographs for the standard are then obtained and peak heights or areas are plotted as a function of conc. A plot of the data should yield a straight line passing thought the origin; analyses are based upon this plot frequent restandardization is necessary for highest accuracy. The most important sources of error in analyses by the method just described is usually the uncertainty in the vol. Of sample, occasionally the rate of injection is also a factor. Ordinarily, sample are small and the uncertainties associated with injection of a reproducible vol. of this size with a micro syringe may amt. to several % relative the situation is exacerbated in gas-liquid chromatography, where the sample must be injected into a heated sample port, here, evaporation from The needle tip may lead to large variation in the vol. injected. Errors in sample vol. can be reduced to perhaps 1% to 2% relative by means of a rotary sample value.

TROUBLE SHOOTING
1.Peaks Broad peaks

Cause Analytes eluted early due to sample overload Detector-cell volume too large

Solution Dilute sample 1:10 and re inject Use smallest possible cell volume consistent with sensitivity needs; use detector with no heat exchanger in system Decrease solvent strength of injection solvent to focus solute; inject smaller volume Use low- or zero-dead-volume end fittings and connectors; use smallest possible diameter of connecting tubing (<0.10 in. i. d.); connect tubing with matched fittings Increase column temperature; change to lower viscosity solvent Decrease injector sample loop size; introduce air bubble in front and back of sample in loop Use smaller-particle-diameter packing, lowerviscosity mobile phase, higher column temperature, or lower flow rate Use gradient elution or stronger isocratic mobile phase Increase sampling frequency Adjust time constant to match peak width

Injection volume too large

Large extra column volume

Mobile-phase solvent viscosity too high

Peak dispersion in injector valve

Poor column efficiency

Retention time too long

Sampling rate of data system too low Slow detector time constant

Ghost peaks Possible Causes Solution

Elution of analytes retained from previous injection

Flush column with strong solvent at end of run; end gradient at higher solvent concentration. Prepare sample in mobile phase; reduce injection volume Prepare trifluoroacetic acid solutions fresh daily; use antioxidant

Ion-pair chromatography - upset equilibrium

Oxidation of trifluoroacetic acid in peptide mapping

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 Cause Solution

Refractive index detection - refractive index of solute Reverse polarity to make peak positive less than that of mobile phase UV-absorbance detection - absorbance of solute less Use mobile phase with lower UV absorbance; if than that of mobile phase recycling solvent, stop recycling when recycled solvent affects detection

Peak doubling Cause Blocked frit Solution Replace or clean frit; install 0.5-um porosity in-line filter between pump and injector to eliminate mobilephase contaminants or between injector and column to eliminate sample contaminants Use sample cleanup or prefractionation; adjust

Coelution of interfering compound

selectivity by changing mobile or stationary phase Coelution of interfering compound from previous Flush column with strong solvent at end of ran; end injection gradient at higher solvent concentration Column overloaded Use higher-capacity stationary phase; increase column diameter; decrease sample amount Replace column, or, if possible, open top endfitting and clean and fill void with glass beads or same column packing; repack column Use weaker injection solvent or stronger mobile phase Use injection volume equal to one-sixth of column volume when sample prepared in mobile phase for injection Replace injector rotor

Column void or channeling

Injection solvent too strong Sample volume too large

Unswept injector flow path

Peak fronting Cause Channeling in column Column overloaded Solution Replace or repack column Use higher-capacity stationary phase; increase column diameter; decrease sample amount

Peak tailing Cause Basic solutes - silanol interactions Solution Use competing base such as triethylamine; use a stronger mobile phase; use base-deactivated silicabased reversed-phase column; use polymeric column See peak doubling Use high purity silica-based column with low tracemetal content; add EDTA or chelating compound to mobile phase; use polymeric column Use polymeric, sterically protected, or high-coverage reversed-phase column; install silica gel

Beginning of peak doubling Chelating solutes - trace metals in base silica

Silica-based column - degradation at high pH

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

Unswept dead volume

Void formation at head of column

Spikes Cause Bubbles in mobile phase Solution Degas mobile phase; use back-pressure restrictor at detector outlet; ensure that all fittings are tight Store column tightly capped; flush reversed-phase columns with degassed methanol

Column stored without caps

2. Leaks Leak at column or fittings Possible Cause Solution

Tighten or replace fitting Catastrophic loose fitting Noncatastrophic white powder at loose fitting Cut tubing and replace ferrule; disassemble fitting, rinse and reassemble.

Leak at detector Possible Cause Solution

Catastrophic detector-seal failure

Replace detector seal or gaskets.

Leak at injection valve Possible Cause Solution

Catastrophic worn or scratched valve rotor

Replace valve rotor

Leak at pump Possible Cause Solution

Catastrophic pump-seal failure

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

3. Recovery Poor sample recovery Possible Cause Solution

Absorption or adsorption of proteins

Change HPLC mode to reduce nonspecific interactions; add protein-solubilizing agent, strong acid or base (with polymeric columns only), or detergent such as SDS to mobile phase.

Adsorption on column packing

Increase mobile phase strength to minimize adsorption; for basic compounds add competing base or use base-deactivated packing Use inert (PEEK), glass-lined, or titanium tubing and flow-path components Ensure no reactive groups are present; use polymeric packing; change column type and mode Use short-chain reversed-phase packing; use 300-A pore diameter packing; use hydrophilic packing or tonexchange media; use hydrophobic interaction chromatography

Adsorption on tubing and other hardware components

Chemisorption on column packing

Hydrophobic interactions between stationary

4. Sensitivity Lack of sensitivity Possible Cause Autosampler flow lines blocked Detector attenuation set too high Solution Check flow and clear any blockages Reduce detector attenuation

First few sample injections - sample adsorption in Condition loop and column with concentrated sample. injector sample loop or column Injector sample loop underfilled Not enough sample injected Peaks are outside detector's linear range response into linear range Sample losses during sample preparation Use internal standard during sample prepartion; optimize sample preparation method See problem: Recovery Overfill loop with sample Increase amount of sample inected Dilute or concentrate sample to bring detector

Sample losses on column

5. Retention Changing retention times

Possible Cause
Buffer retention times

Solution

Use buffer with concentration greater than 20 mM. Contamination buildup Flush column occasionally with strong solvent

Equilibration time insufficient for gradient run or Pass at least 10 column volumes through the column changes in isocratic mobile phase for gradient regeneration or after solvent changes First few injections - active sites Inconsistent on-line mobile-phase mixing Condition column by injecting concentrated sample Ensure gradient system is delivering a constant composition; compare with manually prepared mobile phase; partially premix mobile phase Cover solvent reservoirs; use less-vigorous helium purging; prepare fresh mobile phase Thermostat or insulate column; ensure laboratory temperature is constant.

Selective evaporation of mobile-phase component

Varying column temperature

Decreasing retention times Increasing retention times Possible Cause Decreasing flow rate Solution Check and reset pump flow rate; check for pump cavitation; check for leaking pump seals and other leaks in system. Cover solvent reservoirs; ensure that gradient system is delivering correct composition. Use mobile-phase pH between pH 2 and pH 8

Changing mobile-phase composition

Loss of bonded stationary phase

Retention beyond total permeation volume

6. Equilibration Slow column equilibration time Possible Cause Solution

Reversed phase ion pairing - long chain ion pairing Use ion-pairing reagent with shorter alkyl chain length reagents require longer equilibration time

Varying retention times

Possible Cause
Gradient - insufficient column-regeneration time

Solution
Increase equilibrating time in mobile-phase A to obtain constant retention times for early peaks Increase equilibration time; ion pairing may require as much as 50 column volumes for mobile-phase changeover Pass 10-15 column volumes of mobile phase through column for equilibration

Ion pairing - insufficient equilibration time

Isocratic - insufficient equilibration time

7. Baseline Disturbance at void time Possible Cause Air bubbles in mobile phase Solution Degas or use back pressure restricor on detector

Positive-negative - difference in refractive index of Normal with many samples; use mobile phase as injection solvent and mobile phase sample solvent Drifting baseline Possible Cause Solution

Negative direction (gradient elution) - absorbance of Use non-UV absorbing mobile phase solvents; use mobile-phase A HPLC grade mobile phase solvents; add UV absorbing compound to mobile phase B. Positive direction (gradient elution) - absorbance of Use higher UV absorbance detector wavelength; use mobile phase B non-UV absorbing mobile phase solvents; use HPLC grade mobile phase solvents; add UV absorbing compound to modile phase A.

Noise
Possible Cause Continous - detector lamp problem or dirty flow cell Solution Replace UV lamp( each should last 2000 h); clean and flush flow cell

Gradient or isocratic proportioning - lack of solvent Use proper mixing device; check proportioning mixing precision by spiking one solvent with UV absorbing compound and mointor UV absorbance detector output Gradient or isocratic proportioning - malfunctioning Clean or replace proportioning precision valves; proportioning valves partially remix solvents Occasional sharp interference spikes external electrical Use voltage stabilizer for LC system; use independent electrical circuit Service or replace pulse damper; purge air from pump; clean or replace check valves. Flush column with strong solvent; clean up sample; use HPLC grade solvent Degas mobile phase; use back pressure restrictor at detector outlet

Periodic - pump pulses

Random - contamination buildup

Spikes - bubble in detector

Spikes - column temperature higher than boiling point Use lower column temperature of solvent

8. Pressure Decreasing pressure Possible Cause Insufficient flow from pump Leak in hydralic lines from pump to column Solution Loosen cap on mobile phase reservior Tighten or replace fittings; tighten rotor in injection valve Replace or clean check valves; replace pump seals. Degas solvent; check for obstruction in line from solvent reservoir to pump; replace inlet-line frit

Leaking pump check valve or seals Pump cavitation

Fluctuating pressure Possible Cause Bubble in pump Leaking pump check valve or seals Solution Degas solvent; sparge solvent with helium Replace or clean check valves; replace pump seals

High back pressure Possible Cause Column blocked wth irreversibly adorbed sample Solution Improve sample cleanup; use guard column; reverseflush column with strong solvent to dissolve blockage

Column particle size too small (for example 3 Use larger particle size (for example 5 micrometer) micrometers) 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 Use lower viscosity solvents or higher temperature Replace frit or guard column Replace endfitting or frit assembly

Mobile phase viscosity too high Plugged frit in in-line filter or guard column Plugged inlet frit

Polymetric columns - solvent change causes swelling Use correct solvent with column; change to proper of packing solvent compositionl consult manufacturer's solventcompatibility chartl use a column with a higher percentage of cross-linking Salt precipitation (especially in reversed-phase Ensure mobile phase compatibility with buffer chromatography with high concentration of organic concentration; decrease ionic strength and watersolvent in mobile phase) organic solvent ratio; premix mobile phase When injector disconnected from column - blockage in Clean injector or replace rotor injector

Increasing pressure Possible Cause Blocked flow lines Solution Systematically disconnect components from detector end to column end to find blockage; replace or clean blocked component Filter sample; use .5 micrometer in-line filter; disconnect and backflush column; replace inlet frit Ensure mobile phase compatibility with buffer concentration; decrease ionic strength or water organic solvent ratio

Particulate buildup at head of column

Water-organic solvent systems - buffer precipitation

REFERENCES: http://hydrology1.nmsu.edu/teaching/soil698/Student_Material/HPLCHP1090/hplcuses.htm http://kerouac.pharm.uky.edu/asrg/hplc/applications.html http://en.wikipedia.org/wiki/HPLC http://www.waters.com/waters/nav.htm?locale=en_US&cid=10048919

TECHNOLOGICAL UNIVERSITY OF THE PHILIPPINES TAGUIG CAMPUS 2008-2009

WRITTEN REPORT IN INSTRUMENTATION