What are guard columns and what benefits they offer in HPLC analysis?

HPLC Guard Column

The efficient operation of HPLC system is dependent on freedom of mobile phase and sample from chemical impurities or solid suspensions. Precautions in handling and use of HPLC mobile phase discusses measures that should be adopted during preparation and use of mobile phase. Importance of sample filtration discusses benefits of clean sample injections.

HPLC column is a critical component of the HPLC system which requires careful handling and protection. It is expensive to keep replacing columns frequently so your objective should be to maximize the useful life of the column so that every time you get the desired accuracy and consistency of results.

The chromatographic behavior of the HPLC column begins to decline over use due to gradual accumulation of impurities and suspensions. Particles larger than 2μm present in mobile phase or sample start to deposit on the inlet frit of the column thereby disturbing uniformity of flow. Smaller particles result in increased backpressure so they begin to block the flow path in the stationary phase.

Nature of contaminants

• Highly retained compounds such as fatty acids in reverse phase separations
• Irreversibly retained compounds like residual proteins which were not removed completely at time of sample extraction. Deprotenizatization of proteins in biological fluids before HPLC analysis is strongly recommended.
• Particulate impurities can result from non filtration of samples, particulates released by wear of system components such as seals in the pump or injector.
• Crystalline deposits resulting from drying of residual buffers inside column. Washing of columns with HPLC grade water after use or buffer solutions prevents such salt deposit formation.

What is a guard column?

A guard column is a protective column or cartridge installed between the injector and the analytical column. It serves to remove the impurities and suspended solids from reaching the analytical column. Typically it has a length of about 2 cm and internal diameter of 4.6 mm. Guard columns are packed with pelicullar particles of around 40 μm size to offer negligible pressure drop.

Desirable features of guard columns

• Guard column should have preferably the same packing as the analytical column to eliminate separation complications
• Internal ID of guard column should be comparable to analytical column to minimize back-pressure. Shorter guard column length is preferable but it should be long enough to prevent strongly retained compounds from reaching the main column
• Frit facing the injector should be removable for cleaning by removal of about 2 mm of material and filling with fresh material
• Disposable cartridge type guard columns are convenient and economical to use compared to refillable guard columns.

Guard columns need to be changed on regular basis but intermediate change becomes necessary through observation of changes in chromatographic behaviour such as increase in backpressure, peak broadening and, changes in retention time of peaks. However, the frequency of change can be decided on the basis of chemical composition of sample, presence of highly retained or irreversibly retained components, injection volume or number of injections.

Please share your experiences on use of guard columns and do leave your comments.

Important suggestions for generating reliable HPLC results

Each instrument component contributes to high reliability of results

HPLC has a marked presence in analytical research and quality control laboratories. Important decisions concerning both manufacturing processes as well as publication of research findings are based on decisions which can be arrived at only if high degree of reliability can be placed on the data generated by the HPLC system.

HPLC is a multi component system so the accuracy and reliability of results is dependent on the performance level of each component part. The present article offers some suggestions which will contribute to greater authenticity of results. For simplicity the contribution of each component part is discussed under a separate heading.

Mobile phase

The principal role of mobile phase is to carry the injected sample to the column and after separation in the column to transfer the individual components to the detector for their identification and quantification. Ideally a mobile phase should be prepared fresh before start of analysis and HPLC grade solvents should be used. The solvents should be free of any suspended particles. Initially filter the solvents under vacuum using 0.45 μ filter. Further use of online filters is recommended to remove any residual suspended particles. The mobile phase solvents should be fully miscible with one another and the injected sample should be miscible as well. When using buffers as mobile phase components never allow the buffer to dry out inside the system as salts formed on drying will deposit and cause hindrance to free flow of mobile phase in addition to damage to pump components.

Sample

Like mobile phase solvents it is a good practice to filter the sample before injection. Large volume injections will cause peak shape distortions so columns should not be overloaded by making large volume injections. Whenever a new method is to be developed it is advised to check the compatibility of the sample with the mobile phase. In case the solubility is low try a solvent or combination of solvents matching the polarity of the mobile phase mixture.

Column

 A column is a key component of HPLC system. Its proper care and use will go a long way in providing high reliability of results.

Operating flow rates and pressures should be changed gradually so that such changes do not disturb the stationary phase packing. Use of guard column will also prevent impurities from gaining entry to the main column. As the flow of mobile phase is dependent on temperature use of a column oven is strongly recommended so as to maintain a constant column temperature  for consistency of flow rate.

Pump

The pump is required to provide consistency of flow of mobile phase and maintain the solvent composition throughout the analysis run. In case of gradient analysis a pump is required to maintain the composition of mobile phase as per the software  program. Such conditions are achieved by preventing damage to pump components and timely replacement of such components on noticing any visible damage. Gradually flush the pump by allowing rinsing intermediate polarity solvents or pure water as the case may be.

Injector/autosampler

Consistency of injection volumes is a prime need for reproducibility of results. Manual injectors comprise of fixed volume sample loops and in case of automated modes the injection syringe maintains consistency of volume without operator intervention.

Special care needs to be taken to prevent contamination from previous analysis. In case of manual injectors always flush the injection loop with sufficient sample and in case of automated systems make sure that intermediate wash cycles are part of the analysis program. A validation of the cleanliness of sample and standard reusable vials is vital before commencement of analysis.

Light sources

The majority of HPLC analysis can be carried out using UV light source. Such light sources have a useful life span. Lamp replacement as per recommendations of supplier will contribute to a high degree of reproducibility of absorbance values.

Detectors

A detector is the ultimate part of the HPLC system which confirms the presence of different sample components as well helps in their quantification.

It is recommended that the detector be cleaned periodically from time to time off-line and flushed with water and mobile phase before being re- used. The operating pressure range should never be exceeded to prevent possible damage to detector cell walls.

The suggestions offered in the article will certainly improve the quality of results but it goes without saying that there is no substitute for periodic calibration of the system against reference compounds.

Role of HPLC in Cleaning Validation of Laboratory Glassware

Cleaned and validated glassware ready for use

HPLC has made significant contributions in detection and quantitative estimation of organic compounds. In pharmaceutical analysis HPLC has been extensively used in assay of the active ingredient as well as related impurities which are present in formulations in very low concentrations. Estimation of related substances was not possible using other conventional analysis techniques. This unique ability of HPLC has been successfully exploited for validation of glassware cleaning before it can be put to use for making sensitive analytical determinations.

It is clear that glassware should be cleaned to your complete satisfaction so that you are confident of the results communicated by you. In case you lack confidence then you should be prepared for criticisms on your reports. Cleaning of laboratory glassware suggests cleaning practices for different types of glassware based on the analysis requirements.

Next the question will arise if you can rely on adequacy of your cleaning or further cleaning would be necessary. In other words cleaned glassware will require validation for cleanliness before use. Cleaning validation is a set of practices laid down to ensure the effectiveness and consistency of the cleaning method for removal of residues from earlier analysis, contaminants or even from the detergents used for cleaning. US FDA prescribed screening requirements under GMP through the standard 21 CFR 211.67 covering manufacture and testing of drugs. 

Laboratory glassware cleaning involves repetitive rinses with deionised water or an organic solvent followed by drying in hot air oven. It is important to use water or solvent of high purity grade so that it does not contribute to the contamination of the glassware. During analysis using sophisticated analytical techniques such as HPLC you can come across a set of peaks (referred to as ghost peaks) whose presence cannot be explained. The first thing that can come to your mind could be that such peaks result from impurities present in available standards, blank solutions, reagents or even unclean samples. Such sources of contamination can be investigated independently but due consideration should be given to inadequate cleaning of the glassware before putting it to use.

The residues remaining after cleaning and levels of detergents used in the cleaning process or trace impurities present in de-ionized water or organic solvents necessitates use of a sensitive analytical technique. HPLC is a good choice as it offers a large number of possibilities due to selection from a range of columns and detectors. Generally HPLC systems with a UV detector, photodiode array detector and a C-18 column in reverse phase mode is adequate for the purpose. HPLC offers high sensitivity and detection limits besides providing advanced automation features that can contribute to large scale studies on cleanliness validation of volumetric glassware items as per requirements mandated by the standard.

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

Critical considerations in HPLC solvent selection

Solvent selection for mobile phase preparation requires careful review of its properties

Selection of solvents for use as a mobile phase in HPLC analysis is a key component of method development. It is not possible to hava a universal solvent which will meet all applications and more than often a combination of solvents is decided based on the analysis requirements. Selection of suitable solvents is based on their physical properties and compatibilities with the sample and column stationary phase.

The article discusses some of the essential considerations that play an important role in deciding suitability of solvents as mobile phase.

Cost

Cost is an important consideration as HPLC requires superior purity grade solvents and it is common to see dozens of HPLC systems operating round the clock in large laboratories. This means consumption of high grade solvents in bulk quantities and therefore cost considerations play a vital role.

Solubility

The sample should be completely soluble in the mobile phase. Slightest insolubility will result in phase separations or suspensions which will contribute to operational problems.

Absorbance

Generally the detectors used in HPLC are based on absorbance of light by sample contituents. The intrinsic absorbance of the mobile phase components in the selected wavelength range should not interfere with the absorbance of the sample. The mobile phase solvent should ideally have no absorbance at the wavelength of interest.

Volatility

The mobile phase solvents should have low volatility especially for use with light scattering detectors. Highly volatile solvents can lead to compositional changes in mobile phase composition over use and storage. This can lead to poor reproducibility of chromatograms.

Viscosity

The selected solvents should have low viscosity so that flow through the column does not lead to development of high back pressures.

Inertness

The selected solvents should be inert to sample components, column packing and column material. Any reactivity with any of these components can lead to formation of precipitates, gases or other reaction products which can upset the system performance. The solvents should not form separate phase on coming in contact with the sample. In other words there should be complete miscibility of solvents.

Water

Water is also an important component of reverse phase mobile phase composition either in combination with other solvents or as buffer media. Water purification systems are commercially available. The water generated by such systems should be used at the earliest possible so that its quality does not degrade over the storage period.

HPLC technique is highly sensitive to changes in the composition of mobile phase reaching the detector so it is essential to filter the solvents through 0.45μm filter to remove any solid suspensions. Degassing is also an essential requirement to remove any trace of dissolved air which can lead to flow restriction or spurious peaks.

Local Weather Report and Forecast For: Kakinada Dated :Aug 30, 2020

Local Weather Report and Forecast For: Kakinada    Dated :Aug 30, 2020

Past 24 Hours Weather Data Maximum Temp(oC) (Recorded on 29/08/20) 34.3 Departure from Normal(oC) 2 Minimum Temp (oC) (Recorded. on 30/08/20) 27.6 Departure from Normal(oC) 2 24 Hours Rainfall (mm) (Recorded from 0830 hrs IST of yesterday to 0830 hrs IST of today) NIL Relative Humidity at 0830 hrs (%) 87 Relative Humidity at 1730 hrs (%) (Recorded on 29/08/20) 78 Todays Sunset (IST) 18:15 Tommorows Sunrise (IST) 05:47 Moonset (IST) 03:00 Moonrise (IST) 16:28

7 Day's Forecast Date Min Temp Max Temp Weather 30-Aug 27.0 34.0 Generally cloudy sky with intermittent rain 31-Aug 27.0 34.0 Generally cloudy sky with intermittent rain 01-Sep 27.0 34.0 Generally cloudy sky with intermittent rain 02-Sep 27.0 34.0 Generally cloudy sky with intermittent rain 03-Sep 27.0 34.0 Generally cloudy sky with intermittent rain 04-Sep 28.0 35.0 Generally cloudy sky with possibility of rain or Thunderstorm 05-Sep 28.0 35.0 Generally cloudy sky with possibility of rain or Thunderstorm

                                              

KAKINADA

EXTREME WEATHER EVENTS IN THE MONTH OF AUGUST

Year	Temperature(oC)		Rainfall (mm)
	Highest Maximum(Date)	Lowest Minimum(Date)	24 Hours Highest (Date)	Monthly Total
2019	35.5(17)	24.5(7)	24(14)	108.6
2018	36.3(5)	23.4(9)	29.4(14)	125.7
2017	36.2(2)	24.2(30)	26.9(3)	105.8
2016	36.6(21)	24.2(24)	39.2(24)	53.2
2015	36(25)	24.3(4)	65.6(17)	184.14
2014	36.7(9)	24.5(13)	37(29)	101.1
2013	36(31)	21.7(1)	22(14)	69.4
2012	35.2(17,18)	23.6(4)	55.4(30)	278.8
2011	35.4(13)	23.6(3)	68(3)	232.2
2010	36.4(11)	23(31)	49(13)	241.1
ALL TIME RECORD	38.4(4,7/2009)	21.7(19/1955 & 01/2013)	155.8(08/1983)	374.8(1983)

CLIMATOLOGICAL TABLE

PERIOD: 1981-2010

Month	Mean Temperature(oC)		Mean Total Rainfall (mm)	Mean Number of Rainy Days	Mean Number of days with
	Daily Minimum	Daily Maximum			HAIL	Thunder	FOG	SQUALL
Jan	20.3	29.2	12.6	0.9	0	0.1	0.1	0
Feb	21.7	31.2	10.3	1.1	0	0	0	0
Mar	24.0	34.0	7.5	0.5	0	0.7	0.1	0
Apr	26.2	36.2	16.4	1.1	0	2.1	0	0
May	27.8	37.5	42.3	2.8	0	5.2	0	0
Jun	27.3	35.8	122.8	7.1	0	5.4	0	0
Jul	26.2	32.9	175.4	10.9	0	5.2	0	0
Aug	25.9	32.2	176.9	10.2	0	4.7	0	0
Sep	25.9	32.7	199.4	9.1	0	7.5	0	0
Oct	24.8	31.9	243.4	9.0	0	6.8	0	0
Nov	22.5	30.4	98.8	3.8	0	1.3	0	0
Dec	20.3	29.2	10.7	0.9	0	0	0	0
Annual	24.4	32.8	1116.6	57.5	0	39	0.1	0