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Frequently Asked Questions: HPLC and UHPLC

  1. How much sample can I inject on my LC column and still avoid band broadening?
  2. How much should I change my injection volume if I change the size of my column?
  3. What happens if my sample solvent is stronger than my mobile phase?
  4. Can I get a sharper peak by injecting my sample in a weaker injection solvent (such as 100% water for reverse phase)?
  5. How do I address an increase in backpressure and other signs of blockage or contamination?
  6. How do I determine total column volume or void volume for LC?
  7. Why am I seeing bleed from my Biphenyl column on my UV but not on my mass spec?
  8. What should I use to analyze explosives (as per EPA Method 8330B) by HPLC?

  9. Raptor LC Columns

  10. How does the new Raptor Biphenyl column differ from other Biphenyl columns from Restek?
  11. How exactly does an SPP silica-based column produce higher efficiency and resolution?
  12. Is special conditioning needed for the Raptor Biphenyl column prior to its first use, or if it has been sitting idle?
  13. How much equilibration time is required in between gradient runs on a Raptor Biphenyl column?
  14. What mobile phase solvents are compatible with SPP or Raptor columns?
  15. Can I pump solvent through the Raptor Biphenyl column backwards to clean it?
  16. How much can I inject onto a Raptor column?
  17. How do Raptor ARC-18 columns differ from ordinary C18s?
  18. How well does the Raptor ARC-18 column work for acids and bases?
  19. Can the Raptor ARC-18 column be used with 100% aqueous mobile phases?
  20. How do we know that the Raptor columns are rugged?

  21. Guard Columns

  22. What types of guard cartridge holders does Restek have for HPLC?
  23. Which guard holder configuration is best for my lab?
  24. Is there a Restek guard cartridge to go with my UHPLC column?
  25. What size guard cartridge and holder should I use?
  26. Do I need to use a cap frit with my guard cartridge?
  27. What do I need to purchase to use a Trident integral guard for HPLC?
  28. Can I use a Restek guard cartridge with my column from another vendor?

  29. Hardware

  30. What size threads are on the end fittings of my HPLC column?
  31. What does “10-32” mean?
  32. Are there any other size threads used for LC, besides the 10-32?
  33. How are stainless steel fittings for HPLC different from polymer-based fittings?
  34. Which fittings can be used for UHPLC?
  35. How do I tighten my fittings?
  36. Which fittings do I use for which tubing?
  37. How do I know what the internal diameter of my LC tubing is?

  38. HILIC Methods

  39. How do I avoid problems with HILIC methods?
  40. Is my Raptor HILIC-Si column ready to use right out of the box?
  41. How long should I equilibrate my Raptor HILIC-Si column between injections?
  42. What injection solvent should I use for HILIC separations?
  43. What kind of pH effects do I have to be aware of with HILIC separations?
  44. Can I use buffers for HILIC separations? What kind and what concentration?

If your question doesn’t appear on the list, please contact Restek’s expert chemists, for help troubleshooting HPLC and UHPLC systems and analyses.
(Remember to include your company name and complete mailing address.)

1. How much sample can I inject on my LC column and still avoid band broadening?

Injection volumes, as well as optimal flow rates, are limited by the size of the column. Ideally, if your sample is in the same solvent as your mobile phase, approximate volumes should range as follows:

Column ID Volume (µL)
2.1 mm (30 -100 mm length) 1-3
3.0-3.2 mm (50-150 mm length) 2-12
4.6 mm (50-250 mm length) 8-40

Larger volumes may be acceptable if peaks are still symmetrical. Earlier eluting peaks will exhibit broadening first if the volume is getting too large. If the solvent in your sample is stronger than the mobile phase (starting ratio if using a gradient), you will need to use a smaller volume. Likewise, if your sample solvent is weaker than the mobile phase, you may be able to use a larger volume.

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2. How much should I change my injection volume if I change the size of my column?

Optimal injection volumes are directly related to the cylinder volume of your column and are, therefore, dependent on the cross sectional area (A=π r2) and length (L) of your column. Since that is the case, you can estimate any adjustment from an existing method for injection volume.

If you are converting to a different size ID (with packing material and length remaining the same), just multiply your current volume by the ratio of the radii squared to determine the correct volume for your new method. For example, if you are currently injecting 20 µL on a 150 x 4.6 mm column and then switch to a 150 x 3.0 mm column, you could estimate the adjusted volume by multiplying 20 x (1.52)/(2.32). Your new volume should be about 8.5 µL.

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3. What happens if my sample solvent is stronger than my mobile phase?

We do not recommend injecting in a stronger solvent because it usually results in peak distortion, broadening, poor sensitivity, and shortening of retention times. This happens because some analytes will tend to travel too quickly through the column, instead of eluting in a symmetrical band. If you absolutely must do this, keep the volume as small as possible and make sure the solvents are miscible.

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4. Can I get a sharper peak by injecting my sample in a weaker injection solvent (such as 100%
water for reverse phase)?

In this scenario, the sample is initially concentrated onto the head of the column and moves through the column in a tight band. This technique, referred to as “on-column compression” or “point-injected”, is sometimes used to minimize band broadening for a larger volume sample injection (see FAQ #8 above). Keep in mind that your sample components must be soluble in mobile phase as well, in order for this to work.

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5. How do I address an increase in backpressure and other signs of blockage or contamination?

Please follow the recommendations in our blog “Building up Pressure on HPLC?”. This will help you identify or confirm the source of pressure. If the column itself is found to be the source of difficulty, please follow the column regeneration steps given on our LC Column Cleaning Recommendations page. To help avoid future occurrences, Restek highly recommends the use of guard columns to protect your reversed phase and normal phase analytical LC columns from both frit blockage and column contamination.

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6. How do I determine total column volume or void volume for LC?

The term “column volume” usually refers to the void volume, which represents the volume of mobile phase that is between the silica particles. This area is referred to as the interstitial space. You can estimate void volume by multiplying the total column volume (pi x radius2 x length) by a factor that estimates the typical packing efficiency for a particular column type. For fully porous columns, the equation for void volume (in mL) is V = (0.68) pi r2 L, where V = column volume in mL, r = column radius in cm, and L = column length in cm. For superficially porous columns, such as our Raptor columns, the factor is different and the equation is V = (0.50) pi r2 L.

Void volume is more commonly estimated experimentally by injecting a standard containing an analyte that is known to have no, or negligible, retention on that particular column phase. A good example of this for reversed-phase HPLC is uracil. One should be aware that this estimation is also subject to extra column dead volume for the specific instrument that is being used, so it may vary slightly.

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7. Why am I seeing bleed from my Biphenyl column on my UV but not on my mass spec?

A small amount of phase bleed is inherent for all phases, including phenyl phases, and is somewhat dependent on the size and dimensions of the column. This bleed is usually negligible and does not affect retention times, but may be visible, particularly by UV detection. It can often be reduced after conditioning. Bleed may also be minimized by using an isocratic elution, a shallower gradient, and/or incorporating a gradient flush between runs.

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8. What should I use to analyze explosives (as per EPA Method 8330B) by HPLC?

While no one LC column can provide baseline separation for all of these analytes combined, the Raptor Biphenyl and Raptor ARC-18 columns from Restek are an outstanding choice for primary and confirmation analysis. Fully porous HPLC particles, namely the Ultra C8 and Ultra Aromax columns, are also an option. Keep in mind that a variety of column phases may provide partial solutions for this method, but Restek has found these pairs to give optimal results.

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Raptor LC Columns



9. How does the new Raptor Biphenyl column differ from other Biphenyl columns from Restek?

The Raptor Biphenyl is made with superficially porous particles of silica, often abbreviated as SPP. The SPP silica has a solid core with a porous outer layer and is made with a diameter that is typical of HPLC particles, in this case, 2.7 µm. SPP columns exhibit higher efficiencies and resolution, often approaching that of smaller (such as 1.9 µm) UHPLC particles that are based on fully porous silica. There are also a couple of additional benefits to using solid core particles. SPP silica particles can be operated at high flow rates without significantly impacting column resolution, and they do not require higher pressure system components like UHPLC columns do. (It’s nice to achieve a separation that looks like UHPLC on a regular HPLC system!)

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10. How exactly does an SPP silica-based column produce higher efficiency and resolution?

Greater efficiency is accomplished primarily by shorter transfer time for solutes traveling into and out of the particles and mobile phase, but is also enhanced by more uniform particle size, which results in more consistent packing. From a more theoretical viewpoint, the Van Deemter equation is the best approach to explain this phenomenon. Better efficiency is represented here as a shorter plate height (H).

The Van Deemter equation describes the relationship between column flow rate and peak efficiency, referred to as band broadening.


The solid core of SPP silica results in lower values for the A and B terms (eddy diffusion and longitudinal diffusion) and it results in better mass transfer of solutes, translating to lower values for the mass transfer component, the C term. Therefore, a shorter theoretical plate height H and higher efficiency can be achieved at high flow rates. This is also the case for UHPLC particles, but with SPP particles of normal analytical size, the column pressure is not dramatically higher, as is the case for UHPLC. SPP particles are usually also more uniform in shape and result in more consistent packing, which improves overall efficiency as well. All of these factors produce the significant reduction in band broadening that we commonly associate with SPP silica columns.

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11. Is special conditioning needed for the Raptor Biphenyl column prior to its first use, or if it has been sitting idle?

For the most part, the Raptor Biphenyl column behaves just like any other reversed-phase column. However, in certain circumstances, longer equilibration times may be needed. Switching between organic solvents, such as acetonitrile and methanol, may require a 15-20 minute flush in high organic mobile phase.

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12. How much equilibration time is required in between gradient runs on a Raptor Biphenyl column?

Whether you are using fully porous silica or SPP silica, some equilibration time is needed between runs if you are using a gradient and the amount of time is similar for both types of columns. Usually, the equivalent of 7 column (void) volumes is sufficient unless you are using an ion-pairing technique.

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13. What mobile phase solvents are compatible with SPP or Raptor columns?

Any solvent that is commonly used for reversed-phase LC will work fine, including but not limited to water, methanol, and acetonitrile.

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14. Can I pump solvent through the Raptor Biphenyl column backwards to clean it?

Similar to UHPLC columns, it is not recommended to reverse the flow for these columns. However, you can still pump through a series of solvents, as long as they are miscible.

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15. How much can I inject onto a Raptor column?

Injection volume depends on a number of factors including column dimensions, sample solvents, and analysis requirements. As is always a good practice with chromatography, try to inject as little as possible and in the same or weaker solvent than your mobile phase.

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16. How do Raptor ARC-18 columns differ from ordinary C18s?

The significant difference is the ruggedness of the bonded phase. With the ARC-18, any residual silanol groups are shielded and made inert through steric protection. The result is a wider operating pH range of 1.0–8.0. The ARC-18 is particularly useful between a pH of 1.0 and 3.0, where other C18 column phases may begin to degrade under these harsh conditions. Like the Raptor Biphenyl column, the stationary phase is bonded to superficially porous silica particles (SPP). Please see our earlier FAQ regarding the advantages of SPP.

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17. How well does the Raptor ARC-18 column work for acids and bases?

The ARC-18 provides added retention for charged bases and, in many cases, is preferred over a conventional end-capped C18. For neutral acids, it works well and is preferred over end-capped C18 phases, particularly at pH < 3. The ARC-18 also works for neutral bases and charged acids, but provides more advantages and performs best at the lower pH ranges.

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18. Can the Raptor ARC-18 column be used with 100% aqueous mobile phases?

No. We recommend using the Raptor ARC-18 column with at least 5% organic in the mobile phase. For applications requiring higher aqueous content, we suggest the Ultra Aqueous C18 or Pinnacle DB Aqueous C18 columns.

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19. How do we know that the Raptor columns are rugged?

We use frits that are less prone to clogging from sample matrices, and the column packing is less likely to be damaged by higher pressures that might develop. Added protection of a guard column is also available and recommended to extend the life of the column further. Visit www.restek.com/raptor to see these columns in use and put to the test against the competition, but as the ultimate proof, we encourage you to try them for yourself.

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Guard Columns


20. What types of guard cartridge holders does Restek have for HPLC?

We have 3 styles of guard cartridge holders. They are:

Trident Direct Guard Cartridge System
This has a PEEK tip on one end that screws directly into the column end and is finger-tightened only. The other end is taken apart and tightened with a wrench. That end is removed to insert or change the cap frit and guard cartridge.




Trident In-Line Guard Cartridge Holder

This is the traditional style that is installed by connecting to tubing on either end.





Trident Integral Guard System

This style requires the analytical column to be purchased up front with a unique end fitting. This is indicated for a particular column by adding -700 as a suffix to the catalog number.



More information can be found here:

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21. Which guard holder configuration is best for my lab?

For most applications, we suggest the Trident direct guard, which has low dead volume. It is also easy to use and the style is familiar to many. The Trident integral guard may sometimes be preferred, as it has slightly less volume versus the direct guard. Dead volume becomes more critical with smaller column dimensions and with applications that produce marginal resolution for critical pairs.

The Trident integral guard is purchased by adding the -700 suffix to the catalog number for your column. If you need a guard for an existing column that wasn’t purchased with the Trident integral guard end fitting, there is no way to use the Trident integral guard configuration. The best alternative in that case would be the Trident direct guard system.

If you still find that you need assistance in selecting a guard cartridge configuration, feel free to contact us in Technical Service

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22. Is there a Restek guard cartridge to go with my UHPLC column?

We currently do not carry guard cartridges and holders for UHPLC columns. As an alternative for UHPLC, you may find an in-line filter to be beneficial. We have two types as shown below:



UltraShield UHPLC Precolumn Filter






UltraLine UHPLC In-Line Filter




Similar to our Trident direct guard system, the UltraShield filter screws directly into the column end fitting at the inlet. The UltraLine filter requires a tubing connection on either side.

The other difference is in the frits, or filters as you may call them. The UltraShield unit has a titanium filter which is not replaceable. Therefore, the whole filter assembly is used as a disposable item. On the other hand, the UltraLine filter has a replaceable stainless steel filter and more of them can be ordered as needed.

Filters in both units have a porosity of 0.5 µm and both holder assemblies are leak-tight up to a pressure of 15,000 psi. Therefore, they are functionally equivalent.

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23. What size guard cartridge and holder should I use?

Guard cartridges and corresponding guard holders come in 10 and 20 mm lengths. The 10 mm length works fine for most applications. The 20 mm length is recommended for very dirty sample matrices with many particulates (such as soil and animal tissue) and those that contain reactive compounds (such as derivatizing agents).

Guard cartridges are available in either 2.1 mm or 4.0 mm inner diameter (ID) sizes. Likewise, cap frits are available in 2 or 4 mm frit diameters. You need to choose the size that is closest to the inner diameter of your analytical column, or one size smaller if necessary. The following table provides recommended cartridge and frit dimensions based on the ID of the analytical column.

Analytical Column
ID (mm)
Guard Cartridge
ID (mm)
Cap Frit
Diameter (mm)
1.0 2.1 2
2.1 2.1 2
3.0/3.2 2.1 2
4.0 4.0 4
4.6 4.0 4

Note that the outer diameter (OD) of all guard cartridges is the same regardless of their ID. The same is true of all cap frits, which vary only by the diameter of the stainless steel frit inside the polymer housing.

Be aware that using a guard cartridge that is larger than the ID of your analytical column can result in a loss of efficiency. Also, using a guard cartridge that is more than one size smaller than your analytical column can increase the system backpressure.

We do not offer guard cartridges designed for use with HPLC prep columns.

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24. Do I need to use a cap frit with my guard cartridge?

We recommend placing a cap frit ahead of the guard cartridge because the frit will catch larger particulates and maximize the life of the guard cartridge. Our guard holder systems are designed to work best when a cap frit is present. Porosity is chosen based on the nature of the sample matrix and, in some cases, the nature of the mobile phase mixture. A frit with a porosity of 2 µm can be used in most cases, but a 0.5 µm frit offers more protection for samples that are not pre-filtered or that lack cleanup during sample preparation. Smaller porosity may also be more beneficial when using a 3 µm particle size analytical column or when using a mobile phase that contains higher levels of buffer salts.

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25. What do I need to purchase to use a Trident integral guard for HPLC?


The Trident integral guard cartridges are part of a system of components that must be used together. You will need the following four items in order to use a Trident integral guard for HPLC. Installation instructions can be found here.

  • An analytical column with -700 suffix added to the catalog number is necessary to obtain the proper end fitting. We do not retrofit other existing columns with such an end fitting for this configuration.

  • A guard cartridge that matches your analytical column. Packing material needs to be the same stationary phase (e.g., C18, Aqueous C18, Biphenyl, etc.). Inner diameters of 2.1 and 4.0 mm are available. Particle size for all guard cartridges is 5 µm.

  • An XG-XF fitting to match dimensions of the guard cartridge you plan to use. 10 mm (cat.# 25026) and 20 mm (cat.# 25062) lengths are available.

  • Replacement cap frits are highly recommended.

Please see previous FAQs for selecting length and ID of guard cartridges and cap frits.

If you have an XF end fitting on your analytical column, but decide not to install a guard cartridge/column, please ensure that you remove the XG-XF fitting, otherwise significant dead volume will adversely affect your results. However, you may continue to use a cap frit and keep that in place if you wish.

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26. Can I use a Restek guard cartridge with my column from another vendor?

You can do this for columns with a particle size of 3 µm or greater, provided the packing material is very similar. Some of the column stationary phases/packing materials that are similar to ours are listed in this chart for easy reference: http://www.restek.com/Chromatography-Columns/HPLC-UHPLC-Columns/LC-Columns-Physical-Characteristics-Chart

In some cases, the stationary phase may be so unique that you might need to purchase guard cartridge supplies from the same vendor in order to obtain the best results. Feel free to call us if you have questions about this.

We recommend using only Restek guard cartridge holders with Restek guard cartridges in order to ensure a proper fit.

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Hardware


27. What size threads are on the end fittings of my HPLC column?

Fittings on all HPLC and UHPLC columns have 10-32 threads. However, you will find that fittings and columns from Waters, Rheodyne, SSI, Gasukura have different seating depths, as shown below, even though the threads are still 10-32. Restek’s universal 10-32 PEEK column connectors and new EXP fittings will accommodate any of these configurations.

Fitting styles differ among various manufacturers, but all HPLC and UHPLC column fittings have 10- 32 threads.
 
 
ParkerValcoWatersRheodyneSwagelok
Parker A- Lok

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28. What does “10-32” mean?

This is a naming convention adopted by the American National Standards Institute (ANSI). For HPLC fittings, the first number indicates the diameter of the threaded portion of the nut (not the tubing that goes inside). When the diameter is less than 1/4”, as it is in this case, a gauge value is used (gauge 10). The number following the hyphen refers to the number of threads per inch, thus indicating the “pitch” of the threads. An external screw thread size and tolerances chart is available here: http://www.engineersedge.com/screw_threads_chart.htm


10-gauge fittings are approximately 3/16” in diameter.




Size 10-32 fittings have a thread count of 32 threads per inch. Note that the first thread is used to align the fitting at zero and is not included in the thread count.

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29. Are there any other size threads used for LC, besides the 10-32?

The 10-32 threads are probably the only ones you will use with HPLC. However, you may occasionally see ¼ x 28 or M6 (abbreviated form for M6 x 1) fittings used in laboratory settings or for lower pressure connections. The ¼ x 28 size is based on English measurement units like the 10-32. ¼ x 28 threads are further apart and slightly larger in diameter, and usually they do not withstand higher pressures as well. The M6 fittings are metric threads that are actually 6 mm in diameter and 1 mm apart.

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30. How are stainless steel fittings for HPLC different from polymer-based fittings?

Preseated stainless steel fittings installed on a Restek mixing capillary (cat.# 26533)

The most noticeable difference is that stainless steel fittings are “swaged” onto the tubing. They are tightened with a wrench and once the nut is firmly secured, the ferrule is permanently attached to the tubing. Although this produces a good seal that withstands high pressures, the ferrules cannot be removed and reused. In order to replace a ferrule or reinstall on new tubing, the entire fitting and small section of tubing must be cut, sacrificing the ferrule, as well as a small length of the tubing. (Typically, the nut can still be reused.)

The good news is that polymer-based fittings, such as Restek’s universal 10-32 connectors and hybrid-ferrule EXP fittings, do not require permanent swaging of the tubing; thus, all of the parts can be reused. The EXP fittings are especially useful because they can withstand higher pressures, while the ferrules are still reusable to some degree. The number of times an EXP fitting can be reused is directly related to the amount of torque that is applied. The ferrule can be reused many times when the fitting is finger-tightened, while it can be reused just a few times if the fitting is wrench- tightened for UHPLC. The limitation is due to a certain amount of deformity for the ferrule, not due to swaging.


PEEK finger-tight fittings
( cat.# 25324)

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31. Which fittings can be used for UHPLC?

You can either use the stainless steel fittings that are like the ones that come with your UHPLC system or you can use EXP fittings. The EXP fittings can be used up to 20,000 psi when tightened with a wrench. In any case, always make sure you are using a fitting with zero dead volume so that the high efficiency provided by UHPLC is not compromised by extra dead volume.

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32. How do I tighten my fittings?

Our polymer-based universal connectors and PEEK union connectors only need to be hand-tightened, whereas any of the stainless steel fittings need to be wrench-tightened. The EXP fittings can be used either way. They are hand-tightened for use up to 8,700 psi or wrench-tightened for use up to 20,000 psi. Note that over-tightening causes galling and will destroy the threads. Fittings that need to be wrench-tightened generally require ¼-turn past hand tight to achieve a leak-tight seal. Unfortunately, there is no universal torque setting.

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33. Which fittings do I use for which tubing?


Convention tells us to match fitting material to tubing material: use PEEK fittings with PEEK tubing and use stainless steel fittings with stainless steel tubing. The main reason for that is simply system pressure. However, some stainless steel fittings can be used with PEEK tubing. For example, EXP fittings utilize a titanium/PEEK hybrid construction and can be used with either PEEK or stainless tubing. However, if inertness is a consideration, stainless steel fittings should be avoided and PEEK fittings are a better choice. Please note that PEEK is not recommended for use with some strong acid solutions, such as aqua regia, and it can be affected by halogenated solvents, as well as THF under certain conditions. Stainless steel is safe for most mobile phase reagents and solvents, but it is less inert, particularly toward mineral acids, bases, and oxidizing agents.

PEEK Union

PEEK union connector
(cat.# 27715)

EXP fittings utilize a titanium/PEEK hybrid construction and can be used with either PEEK or stainless tubing.

Other types of tubing, such as PTFE and Tygon tubing, work well with PEEK or polyethylene (PE) fittings, unions, and adaptors. Many of these fittings are not screw-threaded but rather slip-on, because of their use in low pressure applications. A great example of this is mobile phase delivery in HPLC from bottle/reservoir to pump. This is a low-pressure delivery (HPLC and UHPLC systems hold high pressures from pumps to the end of the column), and PTFE tubing and PTFE or PE fittings are commonly used.


 Bluestem glass solvent filter

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34. How do I know what the internal diameter of my LC tubing is?

UHPLC systems are plumbed with extremely narrow-bore tubing that is indiscernible to the naked eye. For this reason, tubing lengths are color-coded based on internal diameter. Restek offers stainless steel capillary tubing with colored bands:
red = 0.005”, yellow = 0.007”, blue = 0.010” and orange = 0.020”. Color coding varies by manufacturer; it is not universal. It’s also important to note that stainless steel capillary tubing should be cut to length by the manufacturer, not by the customer. It is impossible to achieve a clean, square cut without professional machining cutters. Stainless steel fittings with pre-swaged ferrules or EXP fittings with hybrid ferrules are used with stainless steel tubing to achieve leak-tight seals for UHPLC at up to 20,000 psi.

The internal diameter of LC grade stainless steel tubing is identified by the colored band on the pre-cut lengths.

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HILIC Methods


35. How do I avoid problems with HILIC methods?

HILIC methodology is a powerful tool, especially for analyzing polar compounds, but it can be challenging to implement and there are several important considerations that you need to be aware of in order to avoid common pitfalls. Our technical article introduces the HILIC technique, discusses frequent problem areas, and helps you successfully incorporate HILIC methods into your laboratory’s repertoire.

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36. Is my Raptor HILIC-Si column ready to use right out of the box?

Prior to use, your Raptor HILIC-Si column must be properly conditioned with the mobile phase that will be used during analysis. For isocratic HILIC methods, at least 50 column volumes should be used and, for gradient HILIC methods, at least 10 blank injections running the complete time program should be performed. Conditioning is also required when you change the mobile phase composition or the concentration of any additives. Use the same number of column volumes or blank injections as you did when conditioning the column for the first time. Table I below gives the column volumes for Raptor HILIC-Si columns based on their dimensions.

Table I: Column Volumes (mL) Based Upon Raptor HILIC-Si Column Dimensions

 

Column Length

Column ID

30 mm

50 mm

100 mm

150 mm

2.1 mm

0.05

0.1

0.2

0.3

3.0 mm

0.2

0.4

0.5

4.6 mm

0.4

0.8

1.2

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37. How long should I equilibrate my Raptor HILIC-Si column between injections?

In addition to the initial conditioning of the column with the mobile phase, it is critical that the column be re-equilibrated between injections. Because the separation mechanism in HILIC methods involves the adsorption of a water layer on the particle surface, it is very important to completely reestablish or “reset” this water layer in between injections to ensure retention time reproducibility. We recommend equilibration with a minimum of 10 column volumes starting when the gradient program returns to initial conditions, or in isocratic separations, at least 10 column volumes starting from the retention time of the last peak. The number of column volumes required for complete re-equilibration can be very analyte dependent, especially for isocratic separations, so make sure you investigate retention time reproducibility during HILIC method development.

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38. What injection solvent should I use for HILIC separations?

The injection solvent should be as close of a match as possible to the initial mobile phase conditions, which are high in organic content for HILIC separations. By matching the injection solvent to the initial mobile phase conditions, you get better peak shape, increased retention, and higher sensitivity.

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39. What kind of pH effects do I have to be aware of with HILIC separations?

The effect of pH on analyte charge state varies based on each compound’s pKa, so pH effects must be evaluated carefully during method development. With HILIC methods, the high concentration of organic solvent in the mobile phase raises the pH, and the actual eluent pH can be 1–1.5 units higher than in the aqueous portion alone. The charge state of the column itself can also be affected. For example, in a Raptor HILIC-Si column, the bare silica has a pKa between 3.8 and 4.5, so the mobile phase pH changes the charge of the silica surface, making it neutral in very acidic conditions and ionized (negatively charged) as the pH begins to approach 3.8 and above. For this reason, if your analyte has one or more protonated amine or quaternary amine groups, it’s a good candidate for analysis on a Raptor HILIC-Si column.

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40. Can I use buffers for HILIC separations? What kind and what concentration?

Many HILIC separations use a mass spectrometer for the detector, so volatile buffers like ammonium formate and ammonium acetate are very common. However, the high organic content of the mobile phase can cause buffer salts to precipitate, which can lead to downtime for instrument maintenance. In addition, high buffer concentrations can impact chromatography by reducing analyte retention. To avoid these effects, method optimization is required and 10 mM is a good starting point for buffer concentration. Both the A and B mobile phases should be buffered equally in order to keep the ionic strength constant during a gradient for the most consistent MS detector response. Check with your MS vendor for the maximum buffer concentration they recommend for your ESI source.

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