Good Laboratory Practice for HPLC

Preparation of Solvents

Correct solvent preparation is very important. It can save vast amounts of time spent troubleshooting spurious peaks, baseline noise, etc.

A. Quality

• All reagents and solvents should be of the highest quality. HPLC grade reagents may cost slightly more than lower grade reagents, but the difference in purity is marked. HPLC grade reagents contain no impurities to produce spurious peaks in a chromatogram baseline.
• Ensure that any water used in buffer preparation is of highest purity. Deionized water often contains trace levels of organic compounds and so therefore is not recommended for HPLC use.
• Ultra pure HPLC water (18MΩ resistivity) is generated by passing deionized water through an ion exchange bed. Modern water purification instruments use this mechanism to produce water of suitable quality in high volumes. Alternately, HPLC grade water can be purchased from solvent suppliers.

ALSO READ: SOP for Good Laboratory Practices

Important: Do not store HPLC grade water in plastic containers. Additives in the plastic may leach into the water and contaminate it. Always store HPLC grade water in glass containers.

B. Buffers

• All buffers should be prepared freshly on the day required. This practice ensures that the buffer pH is unaffected by prolonged storage and that there is no microbial growth present. Changes in pH and microbial growth will affect chromatography.
• Buffer reagents can contain a stabilizing agent, for example, sodium metabisulphite. These stabilizing agents often affect the optical and chromatographic behaviour of buffer solutions, so it is often worth buying reagents that contain no stabilizer.
• Containers of solid reagent are easily contaminated by repeated use. For this reason, It is recommend that reagents be purchased in low container weights.

C. Filtration

• All HPLC solvents should be filtered through a 0.45 μm filter before use. This removes any particulate matter that may cause blockages.

• After filtration, the solvents should be stored in a covered reservoir to prevent contamination with dust, etc.
• Filtering HPLC solvents will benefit both your chromatography and HPLC system. Pump plungers, seals and check valves will perform better and lifetimes will be maximized.

D. Degassing

• Before the freshly prepare mobile phase is pumped around the HPLC system, it should be thoroughly degassed to remove all dissolved gasses. Dissolved gas can be removed from the solution by

1. Bubbling with helium
2. Sonication
3. Vacuum filtration

• If the mobile phase is not degassed, air bubbles can form in the high-pressure system resulting in problems with system instability, spurious baseline peaks, etc.
• The most efficient form of degassing is bubbling with helium or another low-solubility gas. If this method is available, It is recommended that the mobile phase is continually degassed at very low levels throughout the analysis. This will inhibit the re-adsorption of gases over the analysis time.

Note: Ensure that the solvent reservoir has a vent to the atmosphere to prevent the build-up of pressure inside the reservoir.

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Solvent Use

• Each solvent line should be fitted with an inlet filter. This is the first line of system defense against particulate contamination from solvents.

• The filters 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 lines should be clean, and growth-free, and should have no sharp bends or creases in them. Solvent reservoirs should be placed as high as possible on or in the instrument – always higher than the pump inlet manifold. The solvent lines and filters should be of sufficient length to reach the bottom of the solvent reservoir.

Important: Before starting an analysis, calculate the total volume of the mobile phase required. Prepare all the phases at the same time and place in reservoirs that are large enough to accommodate them. An insufficient mobile phase may cause the system to pump dry. This is undesirable because it will fill the system with air. If this does happen, purge the solvent lines and pump with fresh solvent then allow the system to pump solvent until it is equilibrated.

A. Mobile Phase Properties

• Do not use highly acidic or basic solvents unless your HPLC system and column have been engineered to accommodate them. Seals, plungers, etc. can be damaged by extreme pH conditions.
• The use of highly aqueous mobile phase is becoming more popular as safety guidelines demand less exposure to organic solvents. Care should again be taken that the HPLC column has been engineered to accommodate highly aqueous solvents – traditional alkyl chain media 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 ideal breeding grounds for microbes.
• Ensure that an organic solvent is flushed through the HPLC system and column at least once every 48 hours to kill unwanted microbial growth. Alternatively, add a small amount of sodium azide to the aqueous solvent to inhibit growth.

Note: 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 20% organic in water.

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Changing Solvents

A. Buffered phase to wash 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 change to the proposed wash or storage solvent mix.

B. Normal to reversed phase and Vice versa

• Few columns like Hypercarb can be used in both normal and reversed phase, then with both solvent types.
• To convert a normal phase system/column to a reversed-phase system/column, flush with a solvent that is miscible with both the current normal phase solvents and ideally, the proposed reversed-phase solvents. If the final reversed-phase solvents include a buffer, then it is advisable to move from the 100% methanol flush to a 50% aqueous methanol flush. 
• For example:

1. Normal phase: Hexane/Ethyl acetate
2. Flush: IPA (isopropenyl acetate) then Methanol
3. finally (50:50) Methanol/Water
4. Reversed-phase: Buffered aqueous methanol

• To convert a reversed-phase system/column to a normal-phase system/column, follow a similar path to the one listed previously, but in reverse… For example:

1. Reversed-phase: Buffered aqueous methanol
2. Flush: (50:50) Methanol/Water
3. Methanol then IPA
4. Normal phase: Hexane/Ethyl acetate

C. General

• 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.

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D. Solvent miscibility chart

E. Solvent Properties

The following table list a series of commonly used HPLC solvents and their most pertinent physical properties, including viscosity and miscibility number.

Solvent	Polarity Index	Viscosity (cp) at 20°C	Boiling Point (°C) at 1 atm	Miscibility Number	Refractive Index	UV Cut Off (nm)
Acetic Acid	6.2	1.26	117.9	14	1.372	230*
Acetone	5.4	0.32	56.3	15, 17	1.359	330
Acetonitrile	6.2	0.35	81.0	11	1.344	190
Benzene	3.0	0.65	80.1	21	1.501	280
Chloroform	4.4	0.57	61.2	19	1.443	245
Cyclohexane	0.0	0.90	80.7	20	1.427	195
Dimethyl Sulfoxide	6.5	2.24	189.0	9	1.479	268
p-Dioxane	4.8	1.54	101.3	17	1.422	215
Ethanol	5.2	1.20	78.3	14	1.361	210
Ethyl Acetate	4.2	0.45	77.1	6	1.372	256
Formamide	7.3	3.76	210.5	9	1.446	≈260
Hexane	0.0	0.31	68.7	29	1.372	195
Methanol	6.6	0.60	64.7	11	1.329	205
Methyl Ethyl Ketone	4.3	0.42	80.0	17	1.378	330
1-Propanol	4.3	2.30	97.2	15	1.380	210
2-Propanol	4.3	2.35	117.7	17	1.377	205
1-Propyl Ether	2.4	0.48	91.0	17	1.360	195
Tetrahydrofuran	4.2	0.55	66.0	17	1.407	220
Toluene	2.3	0.59	110.6	24	1.496	285
Water	9.0	1.00	100.0	-	1.333	190
p-Xylene	2.4	0.70	138.0	24	≈1.50	290

*Note: The UV cut-off value for Acetic Acid refers to a 1% solution in water.

​

• 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.
• If the smaller miscibility number is subtracted from the larger 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.
• Solvents have a double miscibility number are immiscible with other solvents at extremes of the lipophilicity scale. The lower of the 2 numbers relates to solvents with high lipophilicity and the second to solvents with low lipophilicity.
• Solvents with double miscibility numbers can, in some circumstances, be immiscible with each other.

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Buffer Properties

• The following table list a series of commonly used HPLC buffers, their alternative name, where applicable, and their pKa values at 20°C.
• Buffer transparency is a variable that should be measured prior to buffer use, as it will vary with salt concentration.
• Buffer choice will be very dependent on the analyte and the instrumentation used. Ideally, LC/MS applications should use a volatile buffer as this will not form a containing deposit on the cone and source.
• Inorganic acids, involatile buffers and ion pair reagents should all be avoided.
• Typical LC/MS buffers include:

1. Ammonium acetate/formate/hydrogen carbonate (< 50 nM)
2. Formic/acetic acid (0.01 – 1% v/v)
3. Trifluoroacetic acid (< 0.1% v/v)
4. Trialkylamine and aqueous ammonia-type bases
5. TRIS (tri-hydroxyméthyl-amino méthane)
6. BIS-TRIS propane

• Electrolyte additives are often added to LC/MS buffers to improve peak shape.
• These additives should also be volatile. Care should be taken when choosing a buffer and additive mixture to ensure that a solution of the two does not produce a solid salt which could cause system contamination.
• Buffers should always be flushed from the analytical column and instrument after use to avoid salts being deposited on delicate frits, etc.

• In reversed phase HPLC, 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.

As acids lose a proton and become ionized (with increasing pH), their retention decreases.

As bases gain a proton and become ionized (with decreasing pH), their retention decreases.

• When separating mixtures containing acids and/or bases by reversed phase HPLC, it is necessary to control the pH of the mobile phase using an appropriate buffer in order to achieve reproducible results.

Example

• A chromatographic separation where resolution goes from an unacceptable 1.4 to an acceptable 3.0 when the mobile phase pH is decreased by only 0.1 units.

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Chromophore Detection Wavelengths

• Chromophores are light absorbing groups. Their behavior is used to allow the detection of analytes. They have one or more detection wavelengths, each of which has a molar absorptivity associated with it. The information contained in the following table is intended as a guide to common chromophores. It is not an exhaustive list.

Column cleaning and regeneration

• In all instances, the volume of solvent used is 40-60 column volumes unless otherwise stated.
• The column efficiency, capacity factor etc…should be measured at the start and end of the clean-up procedure to ensure that it has been performed successfully and has improved the column performance.
• Ensure that no buffers/samples are present on the column and that the solvent used prior to the clean-up is miscible with the first wash solvent.
• After the clean-up, ensure that the test mobile phase is compatible with the last solvent in the column.

A. Normal phase media

• Flush with tetrahydrofuran
• Flush with methanol
• Flush with tetrahydrofuran
• Flush with methylene chloride
• Flush with benzene-free n-hexane

B. Reversed phase media

• Flush with HPLC grade water; inject 4 aliquots of 200 μL DMSO during this flush
• Flush with methanol
• Flush with chloroform
• Flush with methanol

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C. Anion exchange media

• Flush with HPLC-grade water
• Flush with methanol
• Flush with chloroform

D. Cation exchange media

• Flush with HPLC grade water; inject 4 aliquots of 200 μL DMSO during this flush
• Flush with tetrahydrofuran

E. Protein size exclusion media

• There are two wash/regeneration procedures associated with the removal of contaminants from protein size exclusion media.

1. Weakly retained proteins: Flush with 30 mL, 0.1M, pH = 3 phosphate buffer
2. Strongly retained proteins: Flush for 60 minutes using 100% water to 100% acetonitrile gradient.

F. Porous graphitic carbon

• There are four wash or regeneration procedures associated with porous graphitic carbon. The one(s) used will depend on the analytes and solvents that have been used with the column.

Acid/Base regeneration

• Suitable for ionized species analyzed in strongly aqueous mobile phases.
• invert the column
• Flush at 1 mL/min with 50 mL tetrahydrofuran/water (1:1) containing 0.1% trifluoroacetic acid
• Flush at 1 mL/min with 50 mL tetrahydrofuran/water (1:1) containing 0.1% triethylamine or sodium hydroxide
• Flush at 1 mL/min with 50 mL tetrahydrofuran/water (1:1) containing 0.1% trifluoroacetic acid
• Flush with methanol/water (95:5) to re-equilibrate
• Re-invert the column

Strong Organic regeneration

• Suitable for applications involving polar and/or ionized species analyzed in aqueous mobile phases.
• Flush at 1 mL/min with 50 mL acetone
• Flush at 1 mL/min with 120 mL dibutyl ether
• Flush at 1 mL/min with 50 mL acetone
• Flush with aqueous mobile phase until equilibrated

Normal Phase regeneration

• Suitable for applications running predominantly in normal phase mobile phases.
• Flush at 1 mL/min with 50 mL dichloromethane
• Flush at 1 mL/min with 50 mL methanol
• Flush at 1 mL/min with 50 mL water
• Flush at 1 mL/min with 50 mL 0.1 M hydrochloric acid
• Flush at 1 mL/min with 50 mL water
• Flush at 1 mL/min with 50 mL methanol
• Flush at 1 mL/min with 50 mL dichloromethane
• Flush with mobile phase until equilibrated

Removal of Trifluoroacetic acid

• Suitable for applications running mobile phases containing trifluoroacetic acid. Flush the column with acetonitrile that has been heated to 75°C. the column should also be maintained at this temperature.

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 as 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 in 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 PEEK, 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.
• The first table contains information on the compatibility of a wide range of solvents at 20°C with PEEK, polyethylene, polypropylene, PVDF, Teflon® and Tefzel®.
• The second table contains information on the compatibility of PEEK with solvents at elevated temperatures.

1 = Compatible, no adverse effect; 

2 = Application dependant;

3 = Not compatible/recommended.

Band spreading or Band broadening

• Broad peaks, often accompanied by a change in retention time, indicate band spreading. It can occur within the HPLC column, but is more often due to incorrect system plumbing.
• The following procedure describes a method for measuring band spreading due to the HPLC system. Column effects can be measured using efficiency calculations.
• Remove the HPLC column from the system and replace with a zero dead volume union.
• Configure the HPLC system with the following parameters:

1. Flow rate: 1 mL/min
2. Detector sensitivity: 0.5 to 1.0 AUFS
3. Detector time constant: 0.2 or less
4. Chart speed (if required): 20 cm/min

• Perform a ten-fold dilution of the column efficiency test solution. Inject 5 μL of this diluted mix.
• Adjust the detector sensitivity until the peak height approximately 75% of the full-scale readout.
• Measure the peak width at 4.4% peak height (5-sigma column efficiency method).
• convert the peak width to mL using the following conversion:

Band spread (μL) = peak width x (1/20) x 1 x 1000

• Where peak width is measured at 4.4% peak height and expressed in cm; (1/20) represents the chart speed, min/cm; 1 represents the flow rate, mL/min and 1000 represents the volume correction factor, μL/min.

Important: 100 mL ± 30 μL is a typical system band spreading value. Larger values may indicate a problem in the detector, injector, tubing or fittings.

Ensure that your HPLC system does not have built-in extra dead volume, as this will also increase the band spreading value.

• For high values of band spreading, troubleshoot your HPLC system then repeat the determination of band spreading. If the value decreases to acceptable levels, then the problem is resolved. A partial decrease will require further investigation. If the problem persists, contact your instrument supplier for technical advice.
• The table below shows details of the volume of solvent per unit length contained in tubing of varying volumes. For ease of use, both metric and imperial measurements are shown.

Sample preparation

• Sample preparation is about more than just the dissolution of solid in a liquid.
• Samples may require other techniques such as filtration, extraction or derivatization as well as accurate weighing and /or dissolution.

• Samples require filtration if they contain suspended solids. This can be performed on-line using a pre-column filter or as the sample is introduced to the vial.
• Samples that require extraction usually contain the analyte of interest at low levels. The most common forms of extraction are liquid-liquid, solid-liquid and solid phase extraction.
• The latter of these three is perhaps the quickest and easiest to perform. The question most often asked is when do I perform an extraction?
• The simple answer is,Whenever the matrix, that is the substance that your analyte is contained in, is liable to contaminate your HPLC system or block it with particulate matter or
• Whenever the analyte is at such low levels that pre-concentration is required. Solid phase extraction is not difficult to perform and requires very little specialist equipment.
• Sample derivatization can occur before or after the column. It can also be performed manually or automatically. It is generally required for detection purposes, for example, the UV detection of analytes without chromophores.
• There are many different derivatization techniques and care should be taken to choose one that is suitable for your application and that does not produce side products that will cause chromatographic problems later in analysis.

ADDING LC TO MS

• Good Mass Spectrometry will not compensate for Poor Liquid Chromatography. Don’t forget the LC in LC/MS!!!
• Select appropriate chromatography-grade HPLC solvents.
• Avoid exotic solvent mixes MeOH, acetonitrile, water, 0.1% formic acid work best for most of LC/MS applications.
• Dissolve sample in start mobile phase solvent (weakest solvent possible).
• Other solvents: Isopropanol, can be used for normal and reversed phases, Dichloromethane, chloroform, hexane for normal phase with APLCI as ionization.
• Volatile buffers for LC/MS

• Ammonium salts if possible
• Concentrations: 10mM (up to 50mM for APCI), Formic, acetic acids 0.01 – 1% v/v, Trifluoroacetic acid < 0.1% v/v, Alkylamine type bases < 0.1% v/v
• Carbonates are unstable

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