Good HPLC Practices are considered to be Good Laboratory Practices (GLP) and Good Manufacturing Practices (cGMP). A detailed procedure has been given below to maintain High-Performance Liquid Chromatography Instruments.
What are Good HPLC Practices?
The practices which improve Quality and Productivity through the Instrument’s accuracy and reproductive results are also calling as Good HPLC practices. Herein guidance document is providing for better understanding of HPLC analysis.
Important points to remember before starting HPLC analysis
Normal phase chromatography contains a Polar mobile phase and a nonpolar stationary phase.
Correct solvent preparation is very important. It can save the amount of time spent troubleshooting spurious peaks, and baseline noise.
All reagents and solvents should be of the highest quality.
All buffers shall prepare freshly on the same day based on the requirement. All the buffers especially phosphate buffers are bound to microorganisms. Hence, it is always recommended to avoid prolonged storage of buffers.
Filtration & Degassing of HPLC Mobile Phase
Mobile phase preparation, filtration & degassing will play an important role in Liquid Chromatography analysis.
The mobile phase Filtration procedure
All HPLC solvent shall filter through a 0.45 µm unit unless specified. This removes any particulate matter that may cause blockages.
After filtration, the solvents shall store in a covered reservoir to prevent contamination with dust, etc.
Filtering HPLC solvents will benefit both your chromatography and the HPLC system. Pump plungers, seals, and check valves will perform better and maximize life.
Mobile phase Degassing procedure
Before the fresh preparation of the mobile phase for the HPLC system, the solvents shall be thoroughly degassed to remove all dissolved gasses.
Dissolved gas can be removed from the solution by, Bubbling with helium, Sonication, and Vacuum filtration.
If the mobile phase is not degassed, air bubbles can form in a high-pressure system resulting in problems with system instability, spurious baseline peak, etc.
Instrument care for Good HPLC Practices
Inlet filter: Ensure that each solvent line shall fit with an inlet filter. This is the first line of system defense against particulate contamination from solvents. The filter should be kept clean to prevent cross-contamination. When they are not being used, it is recommended that they be stored in a solution of 50% acetonitrile / 50% water. This will inhibit microbial growth and stop dust and dirt from embedding in the filter pores.
The solvent line should be clean, and growth-free and should have no sharp bends or creases in them.
Do not use highly acidic or basic solvents because they will damage mechanical parts (Plunger, seal, etc…) and columns.
Care shall take for HPLC columns as traditional alkyl chains can be prone to phase collapse in low organic composition solvent mixes, for example at less than 5% organic solvent.
Highly aqueous mobile phases are an ideal breeding ground for microbes. After analysis column must flush with a high concentration of the organic mixture.
Never allow an HPLC column or system to stand with water or buffer in it for an extended period of time. Always flush with a solvent mix that contains a minimum of 50% organic water.
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Storage Solvent for HPLC Column & Instrument is Good HPLC Practice
Solvent Changing (Storage Solvent) is an important key aspect of HPLC analysis. The details are given below.
Buffer Phase to rinsing solvent or storage phase
Ensure that the buffer is soluble in the proposed wash or storage phase. If it is not, first flush the system with a solvent mix that is highly aqueous to remove the buffer from the system and column, then the proposed wash or storage solvent mix.
Example: C18 Column with a high concentration of buffer analysis shall store with a high concentration of acetonitrile for storage. A high concentration of acetonitrile cannot introduce inside the column. Because the buffer inside the column and system gets precipitate when acetonitrile mixes with the buffer.
Hence it is always recommended that use the high aqueous solvent mix to remove buffer inside the column and system, then go for a high organic mix for storage of the column/system.
Reverse to Normal Phase
Before removing the solvent bottle ensure that system (All four channels) connected with the union is flushed with water to remove salt/buffer deposited inside the system.
Remove the entire solvent bottle which is used for reverse Phase chromatography, after cleaning of solvent bottle fill nonpolar solvent to be used for Normal phase chromatography for system and column flushing.
Replace the column with the union and put all the channels in the Methanol solvent bottle –give the proportion of each port 25% put flow 2.0 ml/min flush the system for 15 min.
After 15 min keeps the flow at 0.0 ml/min and removes all the channels from Methanol and put it into IPA again continue flushing with 1.0 ml/min (IPA is more viscous than Methanol, hence low flow rate is recommended) for another 30 min.
After 30 min keeps the flow at 0.0 ml/min and remove the entire channel from the IPA solvent bottle and put it into Ethanol again continue flushing with 2.0 ml/min for 15 min.
After 15 min keeps flow at 0.0 ml/min and removes an entire channel from Ethanol to Hexane continue flushing with 2.0 ml/min for 15 min.
Now remove the union and put the respective normal phase column and start column washing with compatible solvent.
Normal Phase to Reverse Phase
Before removing the solvent bottle ensure that system (All four channels) is connected with union flushed with IPA to remove non-polar compound /salt/buffer deposited inside the system.
Flush the system with IPA—-→Methanol—-→Water (method of flushing would be same as in RP to NP)
Caution: Never use water for the Normal Phase column, because silica will form silica gel and destroy the whole column.
Note:
- The normal Phase contains the nonpolar mobile phase and polar stationary phase.
- The reverse Phase contains a polar mobile phase and a nonpolar stationary phase.
- Always refer to relevant standard practices for conversion from one phase to another phase.
Good HPLC Practices & General HPLC Care
Before attempting any solvent change, ensure that the solvent already in the system and column is compatible with the new solvent. If the miscibility or physical properties of the two solvents are unknown, then it is better to mix the solvents in a beaker to see the reaction than to go ahead and pump the second solvent into the first on the HPLC instrument mixing problems are easier to rectify before HPLC.
Solvent Properties and Good HPLC Practices
The following table lists a series of commonly used HPLC solvents and their most pertinent physical properties, including viscosity and miscibility number. Solvent properties will be used during method development for further Good HPLC Practices.
The miscibility numbers can be used to predict the miscibility of solvents. If the smaller miscibility number is subtracted from the larger and the Difference is 15 units or less, then the 2 liquids are soluble in all proportions at 15°C.
In case of the smaller miscibility number is subtracted from the larger one and the difference is 16 units, then the 2 liquids have a critical solution temperature between 25 and 75°C with 50°C as the optimum temperature. If the smaller miscibility number is subtracted from the larger and the difference is 17 or greater, then the 2 liquids are immiscible, or their critical temperature is greater than 75°C.
Effect of pH on Retention time: the retention of analytes is related to their hydrophobicity. The more hydrophobic the analyte, the longer it is retained. When an analyte is ionized, it becomes less hydrophobic therefore its decreases.
System plumbing and fittings
The purpose of a well-plumbed HPLC system is to minimize dead volume between its components and to eliminate leaks. System tubing errors show themselves in many ways, for example, band broadening, baseline noise, etc… detection of incorrect diameter tubing is often very difficult once it is in situ.
The internal diameter of the tubing used in an HPLC system varies with the position of the instrument; refer to your system maintenance manuals.
The type of tubing used is determined by the application that is being performed. The 2 most common types of tubing are steel and PEEKTM, although others are also available. When changing tubing, make sure that the replacement is manufactured from a material that is compatible with any solvents that may be flushed through it. Hence proper fittings will fall under Good HPLC Practices.
Solvent miscibility index
Refer to the below link for Solvent miscibility.
Solvent Compatibility with polymeric tubing
Refer below link for Compatibility.
https://www.bnl.gov/esh/shsd/pdf/compressed_gas/chem_comp_tubing_material.pdf
1= Compatible no adverse effect, 2= Application dependent, 3= Not compatible/ Not Recommended.
HPLC Column flushing before use (Initial usage of HPLC columns)
Column flushing is an important aspect of Good HPLC Practices. The procedure has been elaborated on below.
Reverse Phase Column management for Good HPLC Practices
The column type such as C18, C8, etc, falls under the category of reverse phase. Flush the column, prior to first use for at least 30 minutes using HPLC grade methanol or solvent recommended by the manufacturer at a flow rate of about 1ml/min by ensuring that the flow rate is gradually increased to reach 1 ml/min. Flush the column subsequently, using the filtered, degassed mobile phase of the particular analysis for which the column is issued.
Normal Phase Column management for Good HPLC Practices
The column types such as Silica etc fall under the category of normal phase. Flush the column prior to first use for at least 30 min using HPLC grade n-hexane or solvent recommended by the manufacturer at a flow rate of about 1 ml/min by ensuring the flow rate is gradually increased to reach 1 ml/min. Flush the column subsequently, using the filtered, degassed mobile phase of the particular analysis for which the column is issued.
Regeneration of Column
Column regeneration procedure is available for Reverse Phase HPLC columns and Normal Phase HPLC columns. Following the literature alternatively, the manufacturer’s recommendation can follow for the regeneration of the column. This procedure may or may not be considered Good HPLC Practices based on the Company policy.
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