HPLC - High performance liquid chromatography

High-performance liquid chromatography (HPLC; formerly referred to as high-pressure liquid chromatography), is a technique in analytical chemistry used to separate, identify, and quantify each component in a mixture. It relies on pumps to pass a pressurized liquid solvent containing the sample mixture through a column filled with a solid adsorbent material. HPLC columns Each component in the sample interacts slightly differently with the adsorbent material, causing different flow rates for the different components and leading to the separation of the components as they flow out the column. 

HPLC has been used for manufacturing (e.g. during the production process of pharmaceutical and biological products), legal (e.g. detecting performance enhancement drugs in urine), research (e.g. separating the components of a complex biological sample, or of similar synthetic chemicals from each other), and medical (e.g. detecting vitamin D levels in blood serum) purposes.

Chromatography can be described as a mass transfer process involving adsorption. HPLC relies on pumps to pass a pressurized liquid and a sample mixture through a column filled with adsorbent, leading to the separation of the sample components. The active component of the column, the adsorbent, is typically a granular material made of solid particles (e.g. silica, polymers, etc.), 2–50 micrometers in size. HPLC columns the components of the sample mixture are separated from each other due to their different degrees of interaction with the absorbent particles. The pressurized liquid is typically a mixture of solvents (e.g. water, acetonitrile and/or methanol) and is referred to as a “mobile phase”. Its composition and temperature play a major role in the separation process by influencing the interactions taking place between sample components and adsorbent. These interactions are physical in nature, such as hydrophobic (dispersive), dipole–dipole and ionic, most often a combination.

HPLC is distinguished from traditional (“low pressure”) liquid chromatography because operational pressures are significantly higher (50–350 bar), while ordinary liquid chromatography typically relies on the force of gravity to pass the mobile phase through the column. Due to the small sample amount separated in analytical HPLC, typical column dimensions are 2.1–4.6 mm diameter, and 30–250 mm length. Also HPLC columns are made with smaller sorbent particles (2–50 micrometer in average particle size). This gives HPLC superior resolving power (the ability to distinguish between compounds) when separating mixtures, which makes it a popular chromatographic technique.

The schematic of an HPLC instrument typically includes a sampler, pumps, and a detector. The sampler brings the sample mixture into the mobile phase stream which carries it into the column. The pumps deliver the desired flow and composition of the mobile phase through the column. The detector generates a signal proportional to the amount of sample component emerging from the column, hence allowing for quantitative analysis of the sample components.

A digital microprocessor and user software control the HPLC instrument and provide data analysis. HPLC columns some models of mechanical pumps in a HPLC instrument can mix multiple solvents together in ratios changing in time, generating a composition gradient in the mobile phase.

Various detectors are in common use, such as UV/Vis, photodiode array (PDA) or based on mass spectrometry. Most HPLC instruments also have a column oven that allows for adjusting the temperature at which the separation is performed.

What are preparative, semi-preparative and analytical HPLC?

Analytical HPLC is usually carried out under high backpressures. For example in Reverse-Phase High Performance Liquid Chromatography by using 250 cm length x 4.6 mm Inner Diameter, and 5 micros C18 columns, and flow rates around 0.5 ml/min of solvents like Acetonitrile (low viscosity) and temperatures about 20 or 30oC, HPLC columns the minimum backpressure recommended is 11 bar.

Otherwise, preparative and semi preparative chromatography are carried out under low backpressures (1-5 bar) with bigger inner diameter columns and bigger particle size (10 micros in some cases) by passing mobile phases at high flow rates (4-5 ml/min). Alternatively, you can accomplished preparative and semi preparative chromatography by using a short inner diameter and small size particle (5 micros) by passing a very small flow rate (about 0.05 ml/min) mobile phases with very low viscosity (hexane for example) in the aim of keeping a very low backpressure and saving solvents.

In addition, the most important difference between preparative/semi preparative and analytical HPLC is the purpose. HPLC columns Whereas the first one is designed for obtaining the fractionation of groups of compounds (for example fractionation of PAHs and oxy-PAHs) usually as a function of their polarity, analytical HPLC is designed for separation of individual compounds as a function of their medium polarizabilities.

Maybe these two articles can help understanding the mechanisms of both methodologies.

How can we develop HPLC method for unknown sample

1. First, you should know analytes structure, solubility and choose the detector based on analytes (or your interest) if the analytes are UV active use UV or RID, LSD and MSDetc. HPLC method development start with literature survey to get rough idea about development. HPLC columns start with solubility of the analytes, if it is soluble in polar solvents we can try with reverse phase if it is not we should use normal phase chromatography. Mostly many organic drug molecules are polar or mid polar so we usually go for RPC. 

2. Selection of wavelength by PDA (not always wavelength max, based on related compounds, UV cut off of mobile phase, to avoid matrix interferences etc.) . If the analytes are not UV active then there is dervatisation method.

3. Column selection (start with C18 inertsil and short column (100mm or 150 mm) if it is revesed phase or u can use GL Sciences – Inertsil ODS, waters Xbridge, Agilent Zorbax eclips columns or phenomenex Gemini etc.)

4. Mobile Phase: Gradient setting (Always develop the method by using gradient than isocratic). use buffer or modifier if the analytes are ionisable because ionisable compounds will show as two peaks or split peak as it is existing in two form (HA and A-); HPLC columns so if you buffered the mobile phase into basic or acidic the analyte will go to single form. Maximum use acidic pH (2-4) as the mostly column will hydrolyze more in basic condition

5. HPLC Flush the system with water, check pressure, saturate column with MP, check calibration status, lamp Hours. Always keep data about whatever trials you have done during method development. Method Development is very challenging task so always be logical before any changes in any parameter.

Mostly for HPLC method Development 10 gm sample is enough but for overall development max 100 gm sample is sufficient. It’s a tentative quantity.

How an HPLC/UHPLC Column Protection System Works

Problem: In most UHPLC/ HPLC applications, the buildup of sample and mobile phase contaminants at the head of the chromatography column can cause detrimental effects. 

Particulates may enter the HPLC from many sources, including system wear and tear, sample preparation methods, or from the buffers and solvents used. Sample particulates and contaminants will build up on the column inlet frits or even migrate into the media itself.

Increased system backpressures can result, along with degradation in chromatography, resulting in significantly shortened column lifetimes. Method sensitivity, HPLC columns quantitation and peak identification may all be adversely affected.

When columns have to be replaced more often costs go up, as does system downtime; chromatographers are forced to purchase, install, calibrate and condition a new column, thus impacting laboratory throughput. UHPLC columns packed with sub-2 micron particles tend to clog even more easily and rapidly than traditional HPLC columns packed with larger 3- or 5-micron media. This is because the UHPLC columns have smaller interstitial spaces between particles and the frits used have smaller pores. The restricted flow path makes it easier for particulates to build up, hindering both column and system performance.

Solution: Column protection systems, such as Phenomenex’s SecurityGuard and SecurityGuard ULTRA, can protect columns and detectors from microparticulates from the sample or solvent.

Column protection systems trap particulates and chemical contaminants with inexpensive replaceable cartridges. The cartridges are designed with a short bed length to ensure that they do not alter the chromatography while still protecting the column. Some advanced column protection systems like SecurityGuard ULTRA are specially designed for sub-2 micron, core shell technology HPLC columns (such as Phenomenex’s Kinetex and Aeris) columns and < 3-micron particle columns (< 20,000 psi / 1,378 bar).

SecurityGuard and SecurityGuard ULTRA feature a direct connect cartridge holder with a floating outlet nut that automatically adjusts to the precise port depth of almost every analytical column. This ensures a leak-free and low dead- volume connection, without adapters or couplers. The cartridges, which are packed with matching column media, are designed for easy replacement when the current cartridge is expired.

An accelerated lifetime test, using an endogenous biological matrix injected onto a core-shell column, was conducted and results with and without column protection were compared.

The results clearly showed that sequential injections of the matrix using an unprotected column lead to a steady and irreversible increase in backpressure, where the increase becomes logarithmic. This increase in  backpressure ultimately leads to degraded chromatography, including band broadening and possible peak splitting. System pressure limits may also be quickly reached, at which point the instrument automatically shuts down. Unattended runs may stop prematurely, requiring significant rework by the analyst.

Column lifetime is greatly extended by using the SecurityGuard ULTRA. In this case, sequential injections HPLC columns of the matrix will still lead to an increase in pressure, but this is due to the particulates being captured in the cartridge itself, rather than in the UHPLC/HPLC column. Thus, by simply replacing the cartridge at regular intervals, backpressure returns to starting levels and effective column lifetime can be greatly extended.

What are some of the key factors that affect column performance when working with the new column chemistries?

When using core-shell columns on conventional HPLC, system dead volume contributions from connecting tubing and detector flow cell volume, as well as detector scan rates, need to be addressed in order to attain the full performance benefits that core-shell columns can provide.

Most users will look toward new column chemistries and selectivities when the standard C18 phases do not provide enough resolution for their separation. Typically, one will need to look at the pH of their mobile phase buffers to make sure they are compatible with the new column being tried, as well as the temperature (if performing separations at elevated temperature). In addition, the choice of the HPLC columns organic modifier can have a big impact. For example, with phenyl columns, the pi-pi interactions can be overwhelming when acetonitrile is used. 

Column performance is affected by several factors, including the desired selectivity, the ruggedness and durability of the column packing, and batch-to-batch reproducibility. When working with the newer chemistries, it is most important to read the column manuals and follow the instructions given to achieve optimum results.

What are the Main Benefits of Reversed Phase HPLC?

Reversed high performance liquid chromatography (HPLC) has this name because the order of the process is, as you might expect, reversed. Whereas in normal HPLC the non-polar parts of a substance are separated at the stationary phase, thus eluting the polar ones afterwards, in reverse HPLC the polar ones are subtracted first. This is achieved by using lipophilic groups in the stationary phase, as opposed to the hydrophilic groups in normal HPLC. What this essentially means is that reverse HPLC has the luxury of using water or a water-based solvent in the stationary phase. In normal HPLC, silica is the most commonly-used substance, and while it does have selectivity advantages, it also absorbs water, which can lead to skewed results and retention times. 

What are the Advantages of Reverse HPLC?

By using water (or a water-based substance) as the solvent, reversed HPLC eliminates the danger of the analyte retention times being skewed due to absorption of water in the atmosphere. Moreover, different hydrophobic solvents (commonly used in normal phase HPLC) have differing compression reactions, making the act of accurate gradient separations a much more gruelling task. This is made even harder by the differences in each of their UV cut-off points, as well. With reverse HPLC, the problem is simplified by using more versatile water-based solvents. Furthermore, the traditional way in which the above problems are circumvented using normal HPLC is to employ a more organic solvent than silica; however this often costs more money to dispose of after the procedure is complete. Therefore, reversed HPLC becomes not only simpler, but more cost-effective, too. However, although convenience and economy are two advantages of the process, the main advantage of reversed HPLC  comes in the form of its flexibility. Because it has a hydrophobic stationary phase, it can be used in conjunction with hydrophobic (i.e. non-polar), hydrophilic (i.e. polar), ionic and ionisable compounds in order to separate out their various components, depending on the procedure being used. Quite simply, there is a much vaster choice in which stationary phase to select when it comes to reverse HPLC than there is with normal HPLC.

How to Clean and Regenerate a C8 HPLC Column?

High performance liquid chromatography (HPLC) is a common method of lab analysis in which a sample is separated into its chemical constituents as is passes through a column. The column is filled with a porous packing typically based on treated silica gel. HPLC columns  a standard reverse phase column, such as a C8, can sometimes become clogged or contaminated. Such columns can often be regenerated using a few simple techniques.

Regenerating a Column Detach the fouled column and reverse its position, so that the mobile phase is now entering into what is normally the exit of the column. Attach an exit line to the other end of the column (the end that was originally the inlet), and run this into a waste container.

Pump through a series of solvents in order of decreasing polarity. A basic recommended series is to start with methanol, then progress through acetonitrile, acetonitrile/isopropanol (75:25), isopropanol, methylene chloride and hexane. If the mobile phase originally sitting in the column was a buffer, a wash with water should precede the methanol. A flow rate of 1 to 2 milliliters per minute is typically suitable, HPLC columns  and approximately 10 column volumes of each solvent should be pumped through. The column volume is the internal space not taken up by packing and is calculated as (0.7 x pi x (ID/2)^2 x L), where ID is the column’s internal diameter and L is its length.

Pump another 10 column volumes of isopropanol through the column to prepare it for use with the original mobile phase. If the original mobile phase was buffered, also pump water through the column after the isopropanol before returning to the original buffered mobile phase.

Disconnect the column and reverse and reconnect it so that it is in its normal configuration (that is, with the mobile phase being pumped into the column inlet). Reconnect the outlet of the column to the detector.

Why do we use a Guard Column?

Particulate Contamination

Particles collect in an HPLC column’s inlet frit or in the first millimeter of the packed bed. These valuable columns are usually discarded to avoid degradation of analysis such as peak tailing and split peaks. Instrument downtime can occur which can be very costly to your lab.

Chemical Contamination

Another source of problems are compounds that irreversibly bond to the stationary chromatographic column phase and are often injected into analytical columns. These compounds cause permanent damage to columns that are not protected by a guard column. Shifting of retention time and loss of resolution often results.

A Particulate and Chemical Filter

Particulate contaminants will be filtered by the guard column’s frit before they collect on the analytical column. All dissolved and non-dissolved contaminants will be retained by the guard column’s stationary phase before they destroy the analytical column.

How to Avoid HPLC Column Overload

In High Performance Liquid Chromatography (HPLC), the term overload describes a column condition where a large sample size impairs the performance of the column. Column overload happens when too much sample or solute is injected onto the column. HPLC columns Column overload reduces the performance of the column by causing retention time and peak shape problems. 

Why do columns overload?

A column gets overloaded because there are more solute molecules seeking active sites than there are available sites in the column. In other words, there are a finite number of active sites on the stationary phase of a column available for the solute molecules to transiently bind to. As the sample moves through the column, each solute in the sample will bind to the stationary phase at an appropriate place — as determined by the chromatography conditions.

It is this differential binding that allows the stationary phase to separate the sample into its components.

If all the active sites in the column are taken, then the solute will continue down the column in the mobile phase looking for more active sites. This causes the sample to be spread across a wider area in the column. This can lead to asymmetric peaks and changed retention times.

Of more significance is if overload leads to peak broadening, when small satellite peaks can be subsumed into larger peaks and become undetectable.

Types of overload

There are two main forms of column overload:

Mass overload

Volume overload

The consequences of each of these overload conditions are quite different.

Mass overload

Mass (sometimes termed concentration) overload occurs when too much analyte is injected onto the column. This causes the concentration of the solute in the stationary phase to move away from the idealized behavior represented by the distribution constant. The distribution constant (KC) is the ratio of the concentration of the solute in the stationary phase (CS) to the concentration in the mobile phase (CM). HPLC columns Mass overload results in the concentration range of solute in the stationary phase reaching a non-linear part of the adsorption isotherm.

If the isotherm is concave toward the axis representing the concentration in the mobile phase, at higher solute concentrations of solute, the effective distribution coefficient will be smaller. The higher solute concentrations in the peak will move through the column more rapidly than the lower concentrations and the peak will be distorted with a sharp front and a sloping tail. The overall retention of the solute will be reduced. If the isotherm is concave toward the axis representing the concentration in the stationary phase, then at the higher solute concentrations of solute, the effective distribution coefficient will be larger.

In this case, the high concentrations of solute in the peak will move through the column more slowly than the lower concentrations and the peak will be distorted with a sloping front and a sharp tail. The result is that the overall retention of the solute will be decreased as the mass overload is increased. Thus, peak fronting and peak tailing depend on whether the distribution isotherm curves towards the mobile phase axis or the stationary phase axis.

Volume overload

Volume overload occurs when too much liquid is injected onto the column. Volume overload causes peak broadening but the broadening is symmetrical and so the peak shape at the front and the rear of the peak is not distorted. If an excessively large sample volume is injected onto the column, then the peaks start to tail and the retention times can increase. In volume overload the peak always spreads in one direction towards that of greater retention.

Avoiding overload

Several factors can help avoid column overload:

Samples with a higher concentration of organic solvents than that of the mobile phase should have HPLC columns relatively smaller injection volumes than samples with lower concentrations of organic solvents.

Mass overload tends to start when the sample injection contains above 1 mg of sample per mL of column volume.