Suggestions for trouble free operation of your HPLC system

Regular maintenance is strongly recommended for a smooth and trouble-free operation of all analytical instruments. In addition, proper handling and operation will also contribute to long service life and quality of data obtained from your instruments. The recommendation is even more significant for HPLC as it is one instrument which works like a workhorse round the clock in most of the laboratories.

The article is an attempt to put across some suggestions on the operation of HPLC which will increase its working life and also place high reliability on results of the analysis.

HPLC system

Initial System inspection                          

It is always worthwhile to spend a few minutes before starting analysis on assessing the system suitability for the analysis task in hand. In other words is the system configuration comprising of the pump, injector/autosampler, column, and detector is appropriate for carrying out the analysis in hand. Inspect the tubings and fittings for leak free connections and if necessary replace any worn out connectors.

Filter mobile phase solvents and samples

Always make use of HPLC grade solvents for preparing mobile phases. It is advisable to filter them through 0.22µ size filter prior to mixing. Similarly, it is recommended to filter the samples before introduction into the system.

 Ensure Freedom from air bubbles

It is important to free the delivery lines of any residual air bubbles which can contribute to noisy response and flow restrictions due to the formation of voids in the flow path. Air bubbles can be removed by opening the purge valve and sucking the mobile phase with the help of syringe from the inlet tubing.

Injector

The loop of a manual injector should be flushed with isopropanol to remove any residue from previous samples analyzed. This flushing should be followed by a consistent flow of mobile phase for some time before the introduction of the sample. Similarly, when using the autosampler introduce wash cycles in between sample injections.

Column

The column should be housed inside the thermostatted temperature control oven and should be equilibrated before making the sample and standard injections. In case buffers are part of mobile phase then after completion of analysis flush with water to prevent clogging due to salt formation on drying. When a column is required to be stored flush with original shipping solvent and affix the end caps.

Ensure degassing of the mobile phase to prevent the liberation of air under the high pressures encountered during operation.

Make use of Guard columns

Guard column cartridges fixed before the main column are helpful in extending the useful life of the col impurities.

Start-up and shut down of instrument

On start do not start the system with the final flow rate but increase the flow program in increments of 0.2ml/min steps till the final flow rate is attained.

This will result in minimal disturbance of the column packing. Next program the detector wavelength and injection volume if autosampler facility is to be used. Allow the system to equilibrate till a stable noise-free baseline is obtained. On completion of analysis switch off all the system modules except for the pump. Set the flow rate to 0.2ml/min with the mobile phase followed by the appropriate wash solvent mix until final shutdown. Remove the column and store as recommended if the same column is not required to be used for a considerable time period.

The recommended start-up and shutdown procedure will take a little more time but when adopted as a regular routine it will enhance the useful lifespan of the facility and maintain the reliability and accuracy of results.

HPLC – The new age analytical technique!

HPLC occupies a dominating position in the laboratory

Analytical chemistry is becoming a popular choice as a career option for students pursuing the discipline of chemistry. The obvious reason is that it applies knowledge of chemistry to gain understanding of chemical properties of materials. It makes valuable contribution in establishing the quality of natural resources and synthetic industrial products for our consumption. The quality of such resources and their consistency is greatly dependent on the precise determinations carried out by analytical chemists in their laboratories.

Chromatography in general and HPLC in particular has made remarkable contributions in the areas of research and product development. HPLC deserves special mention as it finds numerous applications in pharmaceuticals, foods, biochemical research, synthetic polymers, studies on bio-molecules, and environmental monitoring. The scope of HPLC is expanding at an astonishing pace and many new applications are being reported in reputed research journals almost daily.

The present article highlights some of the recent developments that have contributed to the large scale acceptance of HPLC as a popular technique in modern day laboratories.

Scale of Operation

HPLC makes it possible to analyze a sample over a vast concentration range and molecular weights. Analysis is conventionally carried out for sample amounts ranging from picograms to gram level quantities through use of different column dimensions and operational modes Developments in field of synthetic polymers and large bio-molecules have resulted from capability of HPLC to handle samples having low molecular weights to those having molecular weights in several millions.

Advances in Stationary Phase Technologies

Silica based columns have remained the backbone of stationary phases to this day. However columns with monolithic phases, zircon base phases and those based on core shell based technologies have contributed to greater speed of analysis, wider temperature and pressure range of operation and studies over extended pH range of media.

Choice of Detectors

HPLC provides several detection options based on the characteristic properties of eluting compounds. The common options available are UV detectors, RI detectors, Photodiode array detectors, Fluroescence detectors, conductivity detectors, light scattering detectors and mass selectivity detectors. LC-MS-MS is today widely acknowledged as a highly sensitive technique which provides analytical solutions in analysis of complex mixtures and low limit detections in bioanalytical studies and studies on pesticides residues in agricultural products.

Choice of Mobile Phases

Reverse phase chromatography is the most popular mode of operation which makes use of polar solvents as mobile phases. However, use of combinations of solvents of different polarities, buffers and isocratic/gradient modes of operation can be useful in resolving complex mixtures of molecules having closely spaced peaks.

The analysis options offered by HPLC technique have contributed to its increasing popularity. Modern day laboratories have several HPLC systems running round the clock. A structured learning of the technique can help contribute value addition to your career growth as an analytical chemist.

Important role of preventive maintenance in smooth functioning of HPLC systems

HPLC manual injector

Preventive maintenance plays a key role in trouble-free operation of laboratory instruments. It is even more critical for instruments that are required to operate round the clock. HPLC is one such instrument which is expected to run continuously in commercial testing laboratories and industries. In pharma and food manufacturing industries quality of products is required to be monitored at regular time intervals and there is need for the system to meet the requirements of online quality control. In case it runs into problems there will be delays in taking decisions on product quality. The same is true in laboratories involved in clinical tests or R&D activities.

Trouble free HPLC operation can be ensured thorough regular maintenance schedules. It may not be possible to follow such schedules on daily basis but if carried out weekly or at regular time intervals they can contribute to significant increase in analysis of samples and loss of valuable time.

The article covers some of the important areas that can be looked into even if the system is running without problems.

Mobile Phase

Mobile phase is the life line of your HPLC

• Use high purity grade solvents, water and reagents for mobile phase preparation
• Filter mobile phase under vacuum using  0.22/0.45μ size filter to remove solid suspensions, if any
• Use online filters in mobile phase containers to make doubly sure that no solid suspended particles reach the column
• Mobile phase should always be degassed to remove dissolved air by sonication, helium sparging or online degassing, if available
• At time of changing mobile phases take out inlet filter from reservoir and put in fresh mobile phase .Open purge valve and purge of the earlier mobile phase completely out of flow line. Close the valve to allow mobile phase to pass to injector and flush it. Finally connect to column to wash it with the new fresh mobile phase
• Use washing solution such as iso-propyl alcohol that is miscible with both previous and next mobile phase. Finally replace intermediate washing solution with fresh mobile phase
• Pass HPLC grade water to wash out buffers. Use of buffers can result in crystalline deposits. The salts can damage plunger or seal and shorten the useful lifetime of the column.

Sample Preparation

• Check solubility of samples in mobile phase before moving to solvents of similar polarity
• Optimize concentration for injection so as to prevent overloading of the column
• Filter sample prior to injection using  0.22/0.45μ size filter

Columns

HPLC columns are expensive and if you take precautions in usage and storage you should get long service from them

• Always avoid overloading the column. For analytical columns the sample load should not be more than 500 μg.
• Avoid sudden changes in operating conditions such as pressure, flow rate, etc
• Always choose guard columns for protection of columns and on changing columns change to recommended guard column
• At end of day remember to carry out washing of columns
• In case column is taken out of system do remember to fix the end caps
• Before storage purge off solid salt deposits and buffers and for long time storage always store column in manufacturer recommended solvent and close the end caps
• Use columns within the pH range 2-8 as columns begin to deteriorate faster outside this range

Pumps

• Operate the system within the recommended flow rates and pressure limits to prevent damage to the seals and O-rings of the pump
• Never leave corrosive solvents or buffers in systems when pump is not running
• Change seals and O-rings periodically as recommended by the supplier. Sonicate inlet filters regularly to prevent any suspended particles from blocking the column
• Prime pump with mobile phase to remove remnants of earlier analysis components
• After using buffer solutions as mobile phases run sufficient volumes of water to remove any salt deposits

Injector

• Flush the injector with mobile phase to remove memory effects and eliminate associated impurity peaks
• Flush with isopropanol in between analysis
• Keep the injector in load position only at time of sample introduction when using a manual injector
• Rinse automated injection systems, needles with water/ isopropanol wash cycles to remove previous sample contamination

Detector

• The detector cell can be cleaned of residues by running water
• Do not exceed the recommended pressure limits to avoid damage to detector cell walls

Column Oven

Column oven maintains constant temperature which prevents drift of retention times of the peaks. The set temperature should be maintained within limits recommended by supplier. Keep the oven clean and periodically check the connections to ensure that there is no blockage of air flow to the oven.

A well maintained HPLC system will give trouble-free operation for continuous operation thereby avoiding breakdowns and help maintain your high sample analysis throughputs.

Restoration of HPLC Column Performance

HPLC columns (Image Courtesy : http://www.knauer.net/)

An HPLC column is capable of giving highly reproducible performance run after run for months together. However, it has a finite lifetime and over a period of time its performance begins to deteriorate as evidenced by distortion of the peak shapes and resolution between them, erratic increase in column back pressure and appearance of ghost peaks. Such abnormalities can be avoided by adoption of recommended restoration practices and care of HPLC columns.

A column can be restored if it becomes fouled due to overloading or use of incompatible mobile phases but a damaged column or one that has out lived its useful life cannot be restored and would require to be replaced.

Column Restoration Procedures

Reverse phase columns

Reverse phase separation is the main operational mode used in majority of HPLC separations. The packings mainly comprise of C18, C4, CN,NH2 and phenyl stationary phases. Eluting with 10 to 20 volumes of solvents in the sequence shown is recommended

• Water
• Acetonitrile
• Isopropanol
• Heptane
• Isopropanol
• Acetonitrile
• Mobile phase

The equilibrium time for common dimensions of HPLC columns are indicated for ready reference :

Column Dimension	Flow rate	Time
250x 4.6mm	1ml/min	60 min
150X 4.6mm	1ml/min	30min

Reverse Phase (Protein/ Peptide separations)

• Flush with 20 volumes of mobile phase without buffer followed by
• 0.1%TFA in water
• 0.1%TFA in acetonitrile/isopropanol(1:2)
• Finally equilibrate with the mobile phase to be used

Normal phase columns

• (Amino, diol, nitro,amino stationary phases)
• Flush with 20 volumes each of
• Heptane
• Isopropanol
• Acetonitrile
• Water
• Isopropanol
• Heptane

Ion Exchange columns

Ion exchange columns comprising of both cation and anion exchangers such as SAX, SCX, WAX, WCX.

• 20 volumes of 500 mM phosphate buffer at pH 7
• 10 percent acetic acid
• 5 volumes of water
• 10 volumes of phosphate buffer at pH 7
• 5volumes of water
• 10 volumes of methanol
• 10 volumes of water
• Mobile phase

GPC/GFC columns

• 5 column volumes of 0.1 phosphate buffer at pH 3
• If strongly retained proteins are used then use a 60 minute gradient from water to acetonitrile to water.

Storage

After regeneration of the column is not to be used immediately then the following storage procedures are suggested :

• Overnight – can be stored in last used mobile phase
• 2-3 days to a week – flush with water to prevent microbial growth
• Long-term storage (several months) – store in aprotic solvents such as acetonitrile.

Validation Before Use

Even after regeneration it is important to validate the performance of a restored column by verifying its performance parameters such as height equivalent to a theoretical plate, asymmetry factor and retention times using standard compound injections.

Advantages of Filtering HPLC mobile phase solvents

HPLC Solvent filtration

HPLC analysis uses liquids as carriers to transport the injected sample to the column and then to the detector. Such carrier can be an aqueous – organic mixture, organic – organic mixture of a buffer in combination with an organic solvent.

It is important to check for complete miscibility of mobile phase solvents before selecting them for any analysis and it is equally important to check that they do not react with each other on mixing. In case the method of analysis is not documented a simple mixing of solvents in a test tube can be helpful in confirmation of both the conditions.

HPLC grade solvents are certified for use in HPLC separations. Even if a small portion of solvent is examined in a beaker it will appear to be crystal clear and suitable for direct use. However, it should be kept in mind that suspended particles or impurities in micron size range are barely seen by the naked eye. Such particles if not removed can in the long run affect the performance of the HPLC system.

Essential precautions in handling and use of HPLC mobile phase will ensure a high level of repeatability and accuracy of analytical results. Bear in mind that water is also an essential component of aqueous mobile phase mixtures. The quality of water used should also conform to the prescribed standards. The earlier article on What is the right quality of water for HPLC analysis is a useful reference.

It is essential to filter the mobile phase components even if you have sourced solvents from reputed suppliers and taken precautions to ensure that buffer salts have been fully dissolved. Suspended particulate matter can result in build up inside columns and result in development of back pressure thereby affecting flow rate and damage to pump components. In the worst scenario there can be mobile phase leakages at joint fittings accompanied with stoppage of the analytical run. Now the question arises on how to minimize such occurrences. Some steps are suggested which will help overcome such situations

• After preparing mobile phase mixture filter through a 0.45 µ size filter using a vacuum
• Prepare mobile phase fresh before use. In case this is not possible every time at least look for any visible suspended impurities or bacterial growth. In such cases it is suggested that the mobile phase should be discarded and another lot freshly prepared
• Use online filters in HPLC solvent bottles to remove any sub- 10 µ size suspended particles. Clean the filters by sonication from time to time
• Filter sample and standard solutions before collection in vials before injection
• Make use of guard columns

The suggested precautions will contribute to the useful life of your HPLC system and at same time improve the reproducibility of your chromatographic separations.