HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

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HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

INTRODUCTION

Basics of the chromatography process

Chromatographic separation process is based on the difference in the surface interactions of the analyte and eluent molecules.

Liquid chromatography is a separation technique that involves:

• The placement (injection) of a small volume of liquid sample

• into a tube packed with porous particles (stationary phase)

• where individual components of the sample are transported along the packed tube (column) by a liquid moved by gravity.

•The components of the sample are separated from one another by the column packing that involves various chemical and/or physical interactions between their molecules and the packing particles.

•The separated components are collected at the exit of this column and identified by an external measurement technique, such as a spectrophotometer that measures the intensity of the color, or by another device that can measure their amount.

There are four major separation modes that are used to separate most compounds:

1. Reversed-phase chromatography

2. Normal-phase and adsorption chromatography 3. Ion exchange chromatography

4. Size exclusion chromatography

(1) Reversed-Phase Chromatography (RPC)

• The column packing is non-polar (e.g. C18, C8, C3, phenyl, etc.) and the mobile phase is water (buffer) + water-miscible organic solvent (e.g. methanol, acetonitrile)

• RPC is, by far, the most popular mode …

• Over 90% of chromatographers use this mode

• The technique can be used for non-polar, polar, ionizable and ionic molecules …

• making RPC very versatile

• For samples containing a wide range of compounds, gradient elution is often used …

• One begins with a predominantly water-based mobile phase and then adds organic solvent as a function of time.

• The organic solvent increases the solvent strength and elutes compounds that are very strongly retained on the RPC packing

(2) Normal Phase or Adsorption Chromatography

• In this mode, the column packing is polar (e.g. silica gel, cyanopropyl-bonded, amino-bonded, etc.) and the mobile phase is non-polar (e.g. hexane, iso-octane, methylene chloride, ethyl acetate)

• Normal phase separations are performed less than 10% of the time.

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• Water-sensitive compounds

• Geometric isomers

• Cis-trans isomers

• Class separations

• And chiral compounds.

(3) Ion Exchange Chromatography

•In ion exchange, the column packing contains ionic groups (e.g. sulfonic, tetra alkyl ammonium) and the mobile phase is an aqueous buffer (e.g. phosphate, formate, etc.).

•Ion exchange is used by about 20%of the liquid chromatographers

•The technique is well suited for:

•The separation of inorganic and organic anions and cations in aqueous solution.

•Ionic dyes, amino acids, and proteins can be separated by ion exchange because such compounds are salt in brine water,

Application Example of Ion Exchange Chromatography Basic proteins on strong cation exchanger (-SO3-) 1. RNA polymerase

2. Chymotrypsinogen 3. Lysozyme HPLC

(4) Size Exclusion Chromatography (SEC)

•In SEC, there is no interaction between the sample compounds and the column packing material. Instead, molecules diffuse into pores of a porous medium. Depending on their size relative to the pore size, molecules are separated. Molecules larger than the pore opening do not diffuse into the particles, while molecules smaller than the pore opening enter the particle and are separated. Large molecules elute first. Smaller molecules elute later

•The SEC technique is used by 10-15% of chromatographers, mainly for polymer characterization and for proteins.

•There are two modes:

•non-aqueous SEC [sometimes termed Gel Permeation Chromatography (GPC)] and

•aqueous SEC [sometimes referred to an Gel Filtration Chromatography (GFC)].

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Column Containing Stationary Phase

Time ^Collect

Components

Load sample Add solvent

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Principles of Liquid chromatography

HPLC is an abbreviation for High Performance Liquid Chromatography (It has also been referred to as High Pressure Liquid Chromatography).

It is the largest separation technique used.

HPLC is a separation technique that involves:

• The injection of a small volume of liquid sample

• into a tube packed with tiny particles (3 to 5 micron (μm) in diameter called the stationary phase)

• Where individual components of the sample are moved down the packed tube (column) with a liquid (mobile phase) forced through the column by high pressure delivered by a pump.

•These components are separated from one another by the column packing that involves various chemical and/or physical interactions between their molecules and the packing particles.

•These separated components are detected at the exit of this tube (column) by a flow-through device (detector) that measures their amount. An output from this detector is called a “liquid chromatogram”.

A typical chromatogram:

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COMPONENTS of HPLC

1. Pump:

• The role of the pump is to force a liquid (called the mobile phase) through the liquid chromatograph at a specific flow rate, expressed in milliliters per min (mL/min).

• Normal flow rates in HPLC are in the 1-to 2-mL/min range.

• Typical pumps can reach pressures in the range of 6000-9000 psi (400-to 600-bar).

• During the chromatographic experiment, a pump can deliver a constant mobile phase composition (isocratic) or an increasing mobile phase composition (gradient).

2. Injector:

• The injector serves to introduce the liquid sample into the flow stream of the mobile phase.

• Typical sample volumes are 5-to 20-microliters (μL).

• The injector must also be able to withstand the high pressures of the liquid system.

• An auto sampler is the automatic version for when the user has many samples to analyze or when manual injection is not practical.

3. Column:

• Considered the “heart of the chromatograph” the column’s stationary phase separates the sample components of interest using various physical and chemical parameters.

• The small particles inside the column are what cause the high back pressure at normal flow rates.

Solvent reservoirs and degassing

5 1

2

4 3 5

Not shown here

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• The pump must push hard to move the mobile phase through the column and this resistance causes a high pressure within the chromatograph.

4. Detector:

• The detector can see (detect) the individual molecules that come out (elute) from the column.

• A detector serves to measure the amount of those molecules so that the chemist can quantitatively analyze the sample components.

• The detector provides an output to a recorder or computer that result in the liquid chromatogram (i.e., the graph of the detector response).

5. Computer:

• Frequently called the data system, the computer not only controls all the modules of the HPLC instrument but it takes the signal from the detector and uses it to determine the time of elution (retention time) of the sample components (qualitative analysis) and the amount of sample (quantitative analysis).

Pump modules

Isocratic pump -delivers constant mobile phase composition;

• solvent must be pre-mixed;

• lowest cost pump

Gradient pump -delivers variable mobile phase composition;

• can be used to mix and deliver an isocratic mobile phase or a gradient mobile phase

•

–Binary gradient pump –delivers two solvents, –Quaternary gradient pump – delivers four solvents

Gradient vs. Isocratic Conditions Isocratic

Mobile phase solvent composition remains constant with time

• Best for simple separations

• Often used in quality control applications that support and are in close proximity to a manufacturing process

Gradient

Mobile phase solvent (“B”) composition increases with time

• Best for the analysis of complex samples

• Often used in method development for unknown mixtures

• Linear gradients are most popular Sample injection

Manual Injector:

1. User manually loads sample into the injector using a syringe

2. And then turns the handle to inject sample into the flowing mobile phase, which transports the sample into the beginning (head) of the column, which is at high pressure

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Autosampler:

1. User loads vials filled with sample solution into the autosampler tray (100 samples) 2. and the autosampler automatically

i. measures the appropriate sample volume, ii. injects the sample,

iii. then flushes the injector to be ready for the next sample, etc., until all sample vials are processed for unattended automatic operation.

HPLC Columns

The separation occurs within the columns Types of columns in HPLC:

•Analytical [internal diameter (i.d.) 1.0 -4.6-mm; lengths 15 –250 mm]

•Preparative (i.d. > 4.6 mm; lengths 50 –250 mm)

•Capillary (i.d. 0.1 -1.0 mm; various lengths)

•Nano (i.d. < 0.1 mm, or sometimes stated as < 100 μm) Materials of construction for the tubing :

•Stainless steel (the most popular; gives high pressure capabilities)

•Glass (mostly for biomolecules)

•PEEK polymer (biocompatible and chemically inert to most solvents) Column packing materials:

Columns are packed with small diameter porous particles.

–The most popular sizes are: 5-μm, 3.5-μm and 1.8-μm

• Columns are packed using high-pressure to ensure that they are stable during use –most users purchase pre-packed columns to use in their liquid chromatographs

• These porous particles in the column usually have a chemically bonded phase on their surface which interacts with the sample components to separate them from one another

–for example, C18 is a popular bonded phase

• The process of retention of the sample components (often called analytes) is determined by the choice of column packing and the selection of the mobile phase to push the analytes through the packed column.

Separation modes of HPLC

The correct selection of the column packing and the mobile phase are the most important factors in successful HPLC.

The two main separation modes used in HPLC are;

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1. Reversed-phase chromatography

2. Normal-phase and adsorption chromatography Detection in HPLC

There are many detection principles used to detect the compounds eluting from an HPLC column. The most common are:

•Spectroscopic Detection

•Refractive Index Detection

•Fluorescence Detection Ultraviolet (UV) Absorption

•An ultraviolet light beam is directed through a flow cell and a sensor measures the light passing through the cell.

•If a compound elutes from the column that absorbs this light energy, it will change the amount of light energy falling on the sensor.

•The resulting change in this electrical signal is amplified and directed to a recorder or data system.

•A UV spectrum is sometimes also obtained which may aid in the identification of a compound or series of compounds.

Variable wavelength detector Diode array detector

Detection Mass Spectroscopy (MS)

•An MS detector senses a compound eluting from the HPLC column first by ionizing it then by measuring it’s mass and/or fragmenting the molecule into smaller pieces that are unique to the compound.

•The MS detector can sometimes identify the compound directly since its mass spectrum is like a fingerprint and is quite unique to that compound.

Refractive Index (RI) Detection

•The ability of a compound or solvent to deflect light provides a way to detect it.

•The RI is a measure of molecule’s ability to deflect light in a flowing mobile phase in a flow cell relative to a static mobile phase contained in a reference flow cell.

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•The amount of deflection is proportional to concentration.

•The RI detector is considered to be a universal detector but it is not very sensitive.

Schematic of a Deflection Type of RI Detector Fluorescence Detection

•Compared to UV-Vis detectors fluorescence detectors offer a higher sensitivity and selectivity that allows quantifying and identifying compounds and impurities in complex matrices at extremely low concentration levels (trace level analysis).

•Fluorescence detectors sense only those substances that fluoresce

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CALIBRATION

Calibration is a comparison between measurements – one of known magnitude or correctness made or set with one device and another measurement made in as similar a way as possible with a second device.

Calibration of an HPLC method is necessary to give quantitative results. However there is more than type of calibration available, and each has its merits and limitations.

There are 5 types of calibration:

1. External Standard Calibration 2. Internal Standard Calibration 3. Area % Calibration

4. Calibration by Standard Addition 5. Calibration using a Correlation Factor 1. External Standard Calibration

This involves analyzing a series of standards covering the concentration range of interest. For example:

Level 1 - 40mg/l Level 2 - 70mg/l Level 3 - 100mg/l Level 4 - 130mg/l Level 5 - 160mg/l

The peak for each component is integrated and identified and the peak area is plotted against concentration to give a calibration curve.

Each component should be present in the standard mix, and hence a series of calibration curves result from a single set of injections, one curve for each component.

When an unknown sample is run, the peaks are integrated and identified, and the peak areas are related to a concentration from the calibration graph.

2. Internal Standard Calibration

An internal standard (IS) calibration is used to eliminate the error caused by variation in injection volume, and to compensate for losses during sample extraction. Injection volume variation is normally very small, but becomes more significant as the injection volume is reduced. An internal standard is an additional component not naturally present in the sample.

A fixed volume is added to all standards and all samples, and instead of plotting peak area against concentration for the calibration curve, we plot:

Peak area of the sample vs. Concentration of the sample Peak area of the internal standard Concentration of the internal standard The principle is that if a smaller or larger injection volume is used, the ratio of the peak areas will be the same, and hence the calibration is valid, even if injection volume errors occur.

3. Area % Calibration

Quantitative results are obtained by adding together the areas of every peak in the chromatogram, and expressing each as a percentage of the total. The results are always given as a percentage, and its primary use is when a species has been purified, and a measure of its purity is required. In this situation, we expect one large peak and a few small ones. The purity

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of the large peak can be read directly from the integration results using Area %. The concentration of the injected sample is not critical – we require only that the main peak is about 90% of full scale to maximize the size of the smaller peaks for ease of integration.

4. Calibration by Standard Addition

A standard addition calibration consists of adding a known amount of the species of interest to the sample, and measuring the increase in detector response. Equating the increase in detector response to the amount of sample added, and assuming linearity of response, we can calculate the original concentration in the sample:

Concentration in original sample, O = SA x A^OS (A^SA- A^OS) where,

SA = Amount of standard added

A^OS= Area of peak without the standard addition A^SA= Area of peak after the standard addition 5. Calibration using a Correlation Factor

The principle is that if it is possible to establish that a linear calibration response is achieved and that the line passes through zero, then the equation for the line is:

y = ax²

For example, there is a fixed correlation factor relating x and y values. Running a single standard concentration we can calculate this correlation factor, and then apply this to all sample peak areas to obtain concentration values.

To use this method, it is essential to confirm linearity by running a series of dilutions which must include a zero blank. Thereafter, it is necessary only to run the single standard periodically during the sequence to confirm that the correlation factor remains constant.

VALIDATION

The route from an idea to an actual standard operating procedure (SOP) may be termed as validation.

Method validation

"Validation of an analytical method is the process by which it is established by laboratory studies, that the performance characteristics of the method meet the requirements for the intended analytical application”

Validation is required for any new or amended method to ensure that it is capable of giving reproducible and reliable results, when used by different operators employing the same equipment in the same or different laboratories. The type of validation programme required depends entirely on the particular method and its proposed applications.

Typical analytical parameters used in assay validation include:

1. Precision 2. Accuracy 3. Linearity 4. Range 5. Ruggedness 6. Limit of detection 7. Limit of quantitation

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8. Selectivity 9. Specificity Precision

The precision of an analytical method is the degree of agreement among individual test results obtained when the method is applied to multiple sampling of a homogenous sample. Precision is a measure of the reproducibility of the whole analytical method (including sampling, sample preparation and analysis) under normal operating circumstances. Precision is determined by using the method to assay a sample for a sufficient number of times to obtain statistically valid results (i.e. between 6 - 1 0). The precision is then expressed as the relative standard deviation.

std dev x 100%

% RSD = ──────────

Mean Accuracy

Accuracy is a measure of the closeness of test results obtained by a method to the true value.

Accuracy indicates the deviation between the mean value found and the true value. It is determined by applying the method to samples to which known amounts of analyte have been added. These should be analyzed against standard and blank solutions to ensure that no interference exists. The accuracy is then calculated from the test results as a percentage of the analyte recovered by the assay. Errors in measurement can be divided into two general categories: systematic errors and random errors;

Systematic errors result from sources that can be traced to the methodology, the instrument or the operator, and affect both the accuracy and the precision of the measurement.

Random errors only affect the precision, and are difficult to eliminate, because they are the result of random fluctuations in the measured signal, due to noise and other factors.

Linearity

This is the method's ability to obtain results which are either directly, or after mathematical transformation proportional to the concentration of the analyte within a given range. Linearity is determined by calculating the regression line using a mathematical treatment of the results (i.e. least mean squares) vs analyte concentration.

Range

The range of the method is the interval between the upper and lower levels of an analyte that have been determined with acceptable precision, accuracy and linearity. It is determined on either a linear or nonlinear response curve (i.e. where more than one range is involved, as shown below) and is normally expressed in the same units as the test results.

Ruggedness

Ruggedness is the degree of reproducibility of results obtained by the analysis of the same sample under a variety of normal test conditions i.e. different analysts, laboratories, instruments, reagents, assay temperatures, small variations in mobile phase, different days etc.

(i.e. from laboratory to laboratory, from analyst to analyst.)

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Limit of detection

This is the lowest concentration in a sample that can be detected, but not necessarily quantitated, under the stated experimental conditions. The limit of detection is important for impurity tests and the assays of dosages containing low drug levels and placebos.

The limit of detection is generally quoted as the concentration yielding a signal-to-noise ratio of 2:1 and is confirmed by analyzing a number of samples near this value using the following equation. The signal-to-noise ratio is determined by:

s = H/h

Where H = height of the peak corresponding to the component

h = absolute value of the largest noise fluctuation from the baseline of the Chromatogram of a blank solution.

The signal (i.e. peak height) can be increased by selecting the optimum monitoring wavelength, increasing the injection volume or mass (below signal or column saturation), increasing the peak sharpness with high efficiency columns and by optimizing the mobile phase. For absorbance detectors, longer path lengths in the flow cell enhance sensitivity though often to the detriment of post column dispersion.

Noise can be reduced by using high sensitivity detectors with low noise and drift characteristics, slower detector response time, mobile phases with low absorbance and pumps with low pulsation.

Limit of quantization

This is the lowest concentration of analyte in a sample that can be determined with acceptable precision and accuracy.

lt is quoted as the concentration yielding a signal-to-noise ratio of 1 0: 1 and is confirmed by analyzing a number of samples near this value (

Selectivity and specificity

Selectivity is the ability to measure accurately and specifically the analyte in the presence of components that may be expected to be present in the sample matrix.

Specificity for an assay ensures that the signal measured comes from the substance of interest, and that there is no interference from excipient and/or degradation products and/or impurities. Determination of this can be carried out by assessing the peak identity and purity.

Diode array detectors can facilitate the development and validation of HPLC assays.

System suitability tests

Once a method or system has been validated the task becomes one of routinely checking the suitability of the system to perform within the validated limits. The simplest form of an HPLC system suitability test involves a comparison of the chromatogram trace with a standard trace.

This allows a comparison of the peak shape, peak width, baseline resolution. Alternatively these parameters can be calculated experimentally to provide a quantitative system suitability test report:

Number of theoretical plates (efficiency) Capacity factor,

Separation (relative retention)

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Resolution, Tailing factor

Relative Standard Deviation (Precision)

These are measured on a peak or peaks of known retention time and peak width.

Plate number or number of theoretical plates (n)

This is a measure of the sharpness of the peaks and therefore the efficiency of the column. The plate number depends on column length - i.e. the longer the column the larger the plate number. Therefore the column's efficiency can also be quoted as:

Either- as the plate height (h), or the height equivalent to one theoretical plate (HETP).

h= L where L = length of column n Or- as plates/meter.

Capacity factor (capacity ratio) k

This value gives an indication of how long each component is retained on the column (i.e. how many times longer the component is retarded by the stationary phase than it spends in the mobile phase).

In practice the k value for the first peak of interest should be >l to assure that it is separated from the solvent.

Separation Factor (relative retention)

This describes the relative position of two adjacent peaks. Ideally, it is calculated using the capacity factor because the peaks' separation depends on the components' interaction with the stationary phase.

Peak Resolution R

This is not only a measure of the separation between two peaks, but also the efficiency of the column. It is expressed as the ratio of the distance between the two peak maxima. (At) to the mean value of the peak width at base (Wb).

Tailing factor T

This is a measure for the asymmetry of the peak.

Relative Standard Deviation or precision

For an HPLC system this would involve the reproducibility of a number of replicate injections of an analytical solution.

The USP requires that unless otherwise specified by a method:

- If a relative standard deviation of <2% is required then five replicate injections should be used - If a relative standard deviation of >2% is required then six replicate injections should be used.

Factors affecting LC system precision

Precision Controlling Factors

Retention time Pump f low and composition precision Column temperature Mobile phase composition

Peak area Autosampler: inj mode, inj volume

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Pump: flow, pulsation

Detector: noise and drift, response Data system: sampling rate,

Integration parameters ANALYSIS

• Separation and analysis of non-volatile or thermally-unstable compounds.

HPLC is optimum for the separation of chemical and biological compounds that are non-volatile.

Typical non-volatile compounds are:

in

• The identification (ID) of individual compounds in the sample.

o The most common parameter for compound ID is its retention time(the time it takes for that specific compound to elute from the column after injection);

o Depending on the detector used, compound ID is also based on the chemical structure, molecular weight or some other molecular parameter.

• The measurement of the amount of a compound in a sample (concentration).

There are two main ways to interpret a chromatogram (i.e. perform quantification):

1. Determination of the peak height of a chromatographic peak as measured from the baseline;

2. Determination of the peak area. In order to make a quantitative assessment of the compound, a sample with a known amount of the compound of interest is injected and its peak height or peak area is measured. In many cases, there is a linear relationship between the height or area and the amount of sample.

• Preparation of Pure Compound(s)

By collecting the chromatographic peaks at the exit of the detector, - and concentrating the compound (analyte) by removing/evaporating the solvent, - a pure substance can be prepared for later use (e.g. organic synthesis, clinical studies, toxicology studies, etc.). This methodology is called preparative chromatography.

• Trace analysis

A trace compound is a compound that is of interest to the analyst but it’s concentration is very low, usually less than 1% by weight, often parts per million (ppm) or lower;

o The determination of trace compounds is very important in pharmaceutical, biological, toxicology, and environmental studies since even a trace substance can be harmful or poisonous;

o In a chromatogram trace substances can be difficult to separate or detect;

o High resolution separations and very sensitive detectors are required.

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PROBLEM SOLVING

Start up - Preliminary checks

Problem Possible cause Solution

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 degassing 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

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

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

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temperature is constant.

Decreasing Retention Times

Active sites on column packing

Use mobile-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

Void Time noise

Air bubbles in mobile phase Degas or use back pressure restrictor 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.

Baseline noise

Continuous - 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 monitor UV absorbance detector output.

Gradient or isocratic proportioning - malfunctioning proportioning valves

Clean or replace proportioning precision valves; partially remix solvents.

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

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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

Decreasing Pressure

Insufficient flow from pump Loosen cap on mobile phase reservoir

Leak in hydraulic 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 pressure

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 with irreversibly adsorbed 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 end fitting or frit assembly

Polymeric columns - solvent change causes swelling of packing

Use correct solvent with column; change to proper solvent compositionl consult

manufacturer's solvent-compatibility chart 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;

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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

Broad peaks

Analytes eluted early due to

sample overload Dilute sample 1:10 and re inject

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 end fittings 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 contamination; use HPLC-grade solvent 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

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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 end fitting 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 saturator column 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 are correctly seated

Void formation at head of column

Replace column, or, if possible, open top end fitting and clean and fill in void with glass beads or same column 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