frequently asked questions about hplc  

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Have a question  about HPLC? It  may have been asked by many other people before. The following frequently asked question list may provide you with a quick answer. If you still cannot find the answer, our specialists may be able to help. We are just one email away.

0. If  I am not sure which column to use and I cannot afford many columns, which column should I start with?  

The most popular column is a C18 (or ODS) column. The column should be able to cover a wide application range, easy to give good result, and has a reasonable price. One example is the PCTsil C18 column
Find out more about a versatile HPLC column.

1. What are the basic components for a HPLC system?  

A workable HPLC system must include a sampler, a pump, a column, a detector, and data processor (computer with software). An degasser and a column oven may also be used for better quality of analysis.  Basically there are two types of designs. One is integrated design which build all the components into one box. The other type is modular design which allows swap and change of components after installation.

2. There are so many brands of HPLC available in the market, how do I judge which one is the best for me?

Although the specification and price are important, they should not be the only standard. For many chemists, the meaning of the specification may not be easy to understand. Sometimes the sales person may mislead you using their specification.  Here are some other items you may consider: a)   feedback from other users, b) hands-on experience or an instrument demo, c) support capability of the vendor, d) cost of maintenance, e) possibility of future upgrade.

3. What are the important specifications for a sampler?

Reproducibility, range of injection volume, linearity over the injection range, and carry over. A good linearity is very helpful when your sample concentration varies a lot or when you want to make a calibration curve using only one or two standard solutions. The carry over property is very critical in trace analysis.   Sometimes a very complicated washing program has to be used before each sample run to avoid carry over  problem. 

4. What are the difference between a high pressure gradient pump and a low pressure gradient pump?

In a low pressure gradient pump, different mobile phase is mixed using a valve before entering the pump head. In a high pressure gradient pump, each different mobile phase is delivered by an individual pump head and then mixed at the pump outlet. The low-pressure gradient design is of lower cost as it only use one pump head. It can also use more types of mobile phase without significant increase of cost. The high-pressure gradient design provides a much faster gradient since solvent mixing point is much closer to the column head. This is measured using delay volume. The value can be 50-300 uL for high pressure gradient pump and can be 2 to 3 times larger for a low pressure gradient pump. A small delay volume is important when the analysis time is short or the flow rate is low. If the delay volume is too large, it become impossible to obtain reproducible gradient run since the planed composition cannot reach the column head before a run is finished..

5. What are the important specifications for an isocratic pump?

Maximum working pressure, flow rate range, flow reproducibility and accuracy, pressure pulsation, pressure monitoring and over pressure shut-off function. 

6. What are the important specifications for a gradient pump?

Maximum working pressure, flow rate range, flow reproducibility and accuracy, gradient reproducibility and accuracy, delay volume,  pressure pulsation, pressure monitoring and over pressure shut-off function.

7. Why is an on-line degasser  always recommended for a gradient pump?

Permanent gas  has different solubility in different solvents. When different solvents are mixed on line, the dissolved gas can be released from solvent due to changed solubility. To verify this, you may add some water to methanol and observe the large amount of bubbles released from methanol. The bubble formed can cause higher baseline nose and shift of retention time.   If you are doing an isocratic LC, a degasser is not so critical since most bubble has been removed when you filter the mobile phase under vacuum. 

8.  How many types of detectors are available for HPLC?

UV detector, fluorescence detector, electrochemical detector, conductivity detector, refractive index detector, evaporative light scattering detector, chiral detector, radioactive detector, mass spectrometry detector (MSD).

9. How to select a suitable detector according to my application?

If your compounds absorb UV light within the range above 200 nm, a UV type detector could be the best choice. It is the most robust and most widely used HPLC detector. Fluorescence detector can have 10 to 100 times better sensitivity for some compounds of large aromatic ring in the structure (such as poly aromatic hydrocarbons). Electrochemical detector is useful for compounds that can be easily electrochemically reduced or oxidized, such as phenols, aromatic amines, and some carbohydrates. It can produce better method sensitivity for such compounds than  a UV detector  due to better selectivity. However, as the electrodes have direct contact with column effluent and involve reaction with sample matrix, the response may not be as stable as a UV detector. Conductivity detector is mainly for inorganic ions in an ion exchange based liquid chromatographic system. Normally a suppressor is needed to remove background ions from mobile phase for a satisfactory detection sensitivity. Refractive index detector can detect any compounds but the sensitivity is around 100-1000 times lower than a UV detector. Another limit of this detector is that it cannot do gradient LC. This detector is mainly used for organic compounds that do not have reasonable UV absorbance, such as small organic acids, carbohydrates, and some polymers. Evaporative light scattering detector is of similar application range as refractive index detector and it can allow gradient HPLC. The disadvantage of this detector is consumption of large amount of nitrogen for evaporating the mobile phase. MSD is getting more and more popular although the price is 10-20  times higher than other HPLC detectors. It can act as a general purpose  detector using scan mode or as an highly selective detector using selective ion monitoring. The most important advantage of MSD is in identity confirmation. 

10. How to choose the flow cell volume for a UV detector?

If the flow cell is too large, two adjacent peaks may get mixed in the cell. If it is too small, the noise may be higher due to less light reaching the photo diodes. The higher back pressure could also limit the flow rate range. The peak size may be used for the selection.  A narrow peak need a smaller flow cell. The rule of thumb is the flow cell volume should not be more than 1/3 of the peak volume. For example,  if the peak width is 0.1 min at 1 mL flow rate, the flow cell volume should not be more than 33 uL. If your flow rate is 0.01 mL/min, the flow cell volume should be around 0.3 uL.

11. How to choose an equivalent C18 column for my HPLC?

The situation in HPLC column is quite different from GC columns. C18 or ODS columns from different supplier can give very different elution profile. This is because the silica properties, the carbon coverage and the surface area varies considerably.  If you want to be sure of the same elution pattern without changing the mobile phase, the column from the same manufacturer should be used. If you want to try a column of similar properties, you can look at columns that have similar surface area and carbon loading for the packing material.

12. How to choose a guard column?

Its volume should not be more than 10% of the analytical column. The packing should be similar to the one in the analytical column. If this is not available, you may use a guard column which has weaker retention to your analytes than the analytical column.

13. What are important specifications for a column?

Separation efficiency, inertness, durability, pH range,  and batch to batch reproducibility.

14. How to choose a chiral column?

It is difficult to predict if a chiral column will give good separation to a pair of chiral isomers. So the best way is to search literature to see if it has been done before. Or you may send your sample to the column supplier for a sample testing.

15. Some columns are end capped. What is it good for?

When the silica is bonded with C18, or C8, there are still some active sites on the silica left over. An end capping using a more active short chain can make the column more inert to some basic compounds and make the column more resistant to basic dissolution to the silica material. However such capping is not stable at low pH (<3). So normally such column is not very suitable for mobile phase of low pH. 

16. How to make a column last longer?

Filter your sample and mobile phase. Make sure the pH of the mobile phase is within the working range of the column. Flush the column with methanol or acetonitrile if it is not going to be used for sometime.

17. Why acetonitrile and methanol are most commonly used solvent for reverse phase HPLC?

These two solvents can mix well with water at any ratio and do not have significant UV absorption within the UV detection range. They are also easy to obtain.  

18. What are the differences between acetonitrile and methanol as mobile phase?

Acetonitrile has lower UV absorbance at short UV region (200-210 nm) and is better choice if your UV detection is in this region (such as for some small organic acids). Acetonitrile also  generate lower back pressure than methanol. This is good when you are trying to use higher flow rate or when your column is partially clogged.  On the other hand, methanol is of lower cost. It is also more stable than acetonitrile. When acetonitrile is dry and is exposed to ambient light, some polymers may be produced which may block your HPLC valves or filters. It is a good idea to use amber bottles for acetonitrile.

19. What are the differences between reverse phase HPLC and normal phase HPLC?

In the reverse phase LC, the mobile phase is polar and the column packing is non-polar. So compounds of high polarity will elute faster than compounds of low polarity. In case of  normal phase HPLC the order  is opposite. The column packing is polar and the mobile phase is of low polarity. Compounds of low polarity will elute faster. Nowadays most people use reverse phase LC due to its better reproducibility and ease in solvent handling. Normal phase LC is easier to have retention time shift due to moisture build up in the silica packing. Normally a gradient elution cannot be used for normal phase LC.  However normal phase LC do have its advantages. It is good for separation of isomers and compounds of very low polarity. Normally reverse phase LC is not good for such situations.

20. Which parameter can tells the instrument status well?

The pump pressure. It reflects the status of column and the system.  A very low pressure (e.g. <10 bar)  indicates leak in the system. A very high pressure tells some block in the column or tubing. If the pressure fluctuate widely (e.g. change from 10 bar to above 50 bar), the pump inlet may have been blocked, some bubble could be in the system,  or the inlet valve is malfunctioning. It is a good habit to keep an eye on the pressure while the instrument is running..


Frequently asked questions about liquid chromatography