HIGH PERFORMANCE LIQUID CHROMATOGRAPHY – HPLC
Here at MA Research product quality is of paramount importance to us. We pride ourselves on being able to put out trustworthy products on a consistent basis. Therefore, all of our products are HPLC tested prior to delivery and come with a 99% purity (or greater) guarantee. So that you can confirm the quality of these products for yourself, HPLC/Mass spec reports are available upon request.
High performance liquid chromatography (HPLC) is an advanced form of column chromatography that is widely acknowledged as one of the most powerful tools in analytical chemistry. It has the ability to separate, identify, and quantitate the compounds that are present in any sample that can be dissolved in a liquid.
Today, compounds in trace concentrations as low as parts per trillion [ppt] are easily identified. Because of this versatility, HPLC is used in a wide variety of industrial and scientific applications, including pharmaceuticals, nutraceuticals, industrial chemicals, environmental matrices, forensic samples, cosmetics, food, and just about anything else you can think of.
How Does It Work?
In simple terms, HPLC determines a mixture’s exact composition by seeing how the individual compounds within that mixture behave in water. It separates and purifies compounds according to their polarity, or their tendency to like or dislike water. To put polarity into context, consider that oil is an apolar liquid that doesn’t mix with water. Ethanol, on the other hand, is polar and as many of you know, mixes very well with water (Vodka and coke anyone?).
I have attempted to simplify the whole process in Figure 1 below, but first let’s look at the main components involved in HPLC.
The HPLC Column
Known as the stationary phase, this is the workhorse of the HPLC machine. It made from one of a variety of substances (most frequently silica), and is very compact in nature. The silica particles are functionalized by long carbon chains. The carbon chains are apolar and therefore the longer the chain, the more apolar the column becomes. Columns containing 18-carbon chains are commonly used and are known as C18 columns.
The HPLC Sample
As mentioned previously, sample types vary greatly and may include pharmaceuticals, nutraceuticals, industrial chemicals, etc.
Injection of Sample
Samples are injected into the HPLC column. This used to be carried out manually, meaning that someone had to sit by the HPLC machine for an extended period of time, often for 8 hours or more, injecting each sample with a syringe.
Fortunately, newer models have an automatic injector, which not only greatly reduces the amount of manual effort involved, but also allows for higher throughput. Modern machines are equipped with software that tells the system what samples to inject, how much to inject, and in what order they should be injected.
The Mobile Phase
This is a mixture of water and an organic solvent (usually acetonitrile or methanol). The mobile phase gets its name because it moves through the column and at the same time elutes (or flushes out) the compounds from the column.
Compounds are often eluted along a concentration gradient. This means that the percentage of water in the mobile phase decreases over time, while the percentage of the apolar solvent increases simultaneously, making the mobile phase gradually more apolar.
The HPLC Run
HPLC can be performed in a number of modes, with the most common being reversed-phase HPLC. This is what is described here. In this mode, compounds are separated beginning with the most polar, and ending with the apolar compounds. For all modes, a high-powered pump moves the sample and the mobile phase through the column. A typical run can take between 10-60 minutes.
The Principle Behind HPLC – A Closer Look
Now that you know the basic components involved, let’s move on to the principle in a little more detail.
I mentioned above that HPLC separates compounds on the basis of polarity, but how does this actually work? As the gradient kicks in, the solvent concentration increases while the water concentration decreases. This makes the mobile phase more and more apolar. Compounds contained in the sample will stick to the carbon chains in the column, with the most apolar compounds sticking the strongest, and the most polar compounds sticking weakly.
Figure 1 shows what happens to a sample containing a mixture of compounds after injection into the column. The compounds bind to the column and are flushed out at different times, depending on whether they are more likely to stick to the column or the mobile phase as it is pumped through. The time that each compound elutes (or flushes out) from the column is known as that compound’s retention time.
Figure 1: Compounds of differing polarities (indicated as darkening shades of blue) are injected into the HPLC column (entire cylinder). The mobile phase is pumped through the column, and the addition of solvent along a concentration gradient (shown as a black dotted line) continuously decreases the overall polarity of the mobile phase (Y-axis). Compounds are able to stick to either the column or the mobile phase, depending on how polar they are. Compounds will eventually stick to the mobile phase when their polarity matches that of the mobile phase. They will then dissociate from the column and will be eluted at a particular time (X-axis) during the run. This time is known as the Rf for that compound.
Understanding the Output
The output or results of an HPLC run is usually viewed as a chromatogram (Figure 2). This is a horizontal series of peaks representing compounds eluted from the column with different Rf values. Modern HPLC equipment is often coupled to a diode array detector (DAD), allowing the user to look at the resulting chromatogram of separated compounds in wavelengths from 190 nm to 900 nm. If the compounds under investigation are known, the user can choose to look only at 1 or a few selected wavelengths. For instance, cocaine can be observed at 254 nm.
Figure 2: This chromatogram shows the separation of compounds from a chemical reaction, and the chromatogram is viewed at 254 nm. Two main peaks occur at 8.20 and 9 minutes, representing two compounds with these retention times. The number of absorbance units (AU) is shown on the Y-axis while the time of the run is shown on the X-axis.
Within biology and medicine, HPLC is often used as an analytical tool to assay biological and environmental samples for the presence or absence of known compounds (for example metabolites, drugs, toxins, pesticides), and can assist in the identification of unknown compounds.
Within chemistry, however, HPLC is routinely used to monitor chemical reactions, as well as to determine the purity of the products. In addition, the process of HPLC can be modified to preparative HPLC, whereby compounds of interest can be purified for further use.
While the vast majority of MA Research products are made within the U.S.A and 3rd party HPLC/MASS spec tested, a very limited number of chemicals, due to difficulty in sourcing and/or regulations surrounding their production and/or purchases made in conjunction with other companies/individuals, prevents us from manufacturing all of our products within the U.S.A. As a result, some of these chemicals have been subject to lab testing prior to MA Research assuming possession, the results of which have been accepted in good faith due to the long-standing and close nature of these on going business relationships.