USP Guideline Note: HPLC Column Diameter Changes to Maintain Flow Rate Linear Velocity

USP Allowed Variations in HPLC Column Diameter (*USP 32, Second Supplement, Dec 1, 2009). In the previous USP version, a change of up to 50% of the flow rate was allowed. This has been changed in the more recent version. A wide range of column diameter changes are now allowed, provided that the linear velocity is kept constant. *We addressed the effect of changing column diameter on flow rate in a previous blog post, but this time I have also expanded on the calculation by including the extra variable for column length (L1 and L2) as well.

*Adjusting the Column Flow Rate for Changes in the HPLC Column Diameter.

Linear Velocity Formula:

   New Linear Flow Rate₂ = Flow Rate₁ x (L2 x D2²) / (L1 x D1²)

Flow Rates are in ml/min.

L1 = Column Length (original) in mm.

L2 = Column Length (proposed) in mm.

D1 = Column Diameter (original) in mm.

D2 = Column Diameter (proposed) in mm.

Example #1:

Original column is a 150mm x 4.6mm (L x ID) used at 1.000 ml min. We would like to determine what the equivalent flow rate (F₂) would be for a column which is 150mm x 2.1mm (L x ID) to maintain the same linear velocity. This is a proposed change in column diameter of > 50% so it would not have been allowed under the old guidelines. The newer guidelines take into account that with the same particle size, changing the column diameter will not change the chromatography if the linear velocity is maintained as before. Let’s calculate the new flow rate using the formula above.

1.000 x (150 x 2.1²) / (150 x 4.6²) = F₂

                    1 x (661.50 / 3,174) = F₂

                              0.208 ml/min = F₂

Popular LC/MS and HPLC Volatile Mobile Phase Modifiers

For applications which utilize an Evaporative Light Scattering Detector (E.L.S.D.), Charged Aerosol Detector (CAD) and/or Mass Spectrometer Detector with Electrospray Ionization source (e.g. LC/MS, MSD or LC/MS/MS), a fully volatile buffering system is usually required. Many of the common HPLC buffers such as sodium or potassium phosphate are not compatible.Use the smallest amount of buffer which provides buffering under the analysis conditions (e.g. 10mM). *Select a buffering agent (or modifier) which are within 2 pH units (+/- 1) of the sample's pKa and 2 pH units away from any acid's pKa. 

• For LC/MS applications: Positive ion mode favors acidic mobile phases and Negative ion mode favors basic mobile phases. However, feel free to experiment using both ionization modes and don't forget about using adducts (e.g. ammonium and sodium) with all types of samples to improve signal response. *Maintain these buffers at or below 10 mM. Adjust the pH of the mobile phase to be 1 to 2 units away from your sample's pKa.

Table 1:  Popular examples of useful volatile mobile phase buffers, modifiers and/or additives.

BUFFERING/MODIFIER AGENT                                   USEFUL pH RANGE

• Ammonium formate                                 2.8 - 4.8; 8.2. - 10.2
• Formic Acid                                            3.3 - 4.3
• Pyridine/Formic Acid                               3.3. 4.3, 4.8 - 5.8
• Trimethylamine/Formic Acid                     3.3 - 4.3, 9.3 - 10.3
• Ammonium Acetate                                  3.8 - 5.8; 8.2 - 10.2
• Acetic Acid                                              4.3 - 5.3
• Trimethylamine/Acetic Acid                      4.3 -5.3, 9.3 - 10.3
• Ammonia/Formic Acid                              3.3 - 4.3, 8.8 - 9.8
• Ammonia/Acetic Acid                               4.3 - 5.3, 8.8 - 9.8
• Ammonium Bicarbonate                           5.9 - 6.9,  8.8 - 9.8
• Ammonium Carbonate                              5.9 - 6.9, 8.8 - 9.8  
• Carbonic Acid                                            6 - 8 (pKa 6.37/pKb 7.63)
• 1-Methylpiperidene                                   10.0 - 12.0  

• Trifluoroacetic Acid (TFA)                        pKa = 0.3 (WARNING when used with MS

                                                                                     systems!).  See notes #2 and #4 below.           

*Notes: (1) Formic acid (3.75) is slightly stronger and more volatile than Acetic acid (4.75). Formic acid is often available in higher purity grades and absorbs less in the UV region making it a better choice for most chromatography applications. It works well in positive mode LC/MS analysis, esp at 0.1%. (2) Trifluoroacetic acid (TFA, pKa = 0.3) is very strong and volatile, but we do not recommend its use in LC/MS applications as it can increase the background signal levels (esp. in Negative Mode) LC/MS (m/z 113), be very hard to remove from the source and result in long term instrument contamination. Difluoroacetic acid (DFA) and ammonium formate are other alternatives as they offer good ion pairing capacity with less ion suppression problems. (3) Triethylamine (TEA, pKa 11) is volatile, strong and very stable, but causes similar contamination problems resulting in high background signals when used in Positive Mode LC/MS (m/z 102). (4) Many ion-pairing reagents suppress ionization, bind to the plastics and metals used and contaminate the flow path. If you must use them, please do so using the lowest possible concentrations levels and thoroughly decontaminate the entire flow path of the system after use (or dedicate the MS system to use with them only). Minimize further contamination by labeling and using a dedicated column for the application (Do not use that same column exposed to ion pairing compounds for any other methods or applications). (5) Acids and bases alone provide little "buffering" so should be used with a secondary buffering species to resist change in pH.

Gradient Mixing Test For Your HPLC Pump (Step Gradient)

The most popular type of gradient pumping module used to perform HPLC analysis utilizes a low pressure mixing valve in their design. These valves are electronically controlled and proportion the amount of mobile phase from one of several solvent channels into a mixer for introduction to the pump head (*the solenoid valves used for this are sometimes called gradient proportioning valves). They provide random access to multiple solvents (e.g. 4) for method development and column flushing. The mobile phase solutions are mixed at low pressure before entering the high pressure side of the pump head (where they undergo compression). This design requires only one high pressure pumping head and can allow for very high mixing accuracy (often 0.1% per channel) of the mobile phase. This allows for the formation of mobile phase gradients over time which greatly aid in resolving samples apart on the column.

The gradient proportioning valves need to be tested along with the other parts of your HPLC system on a regular basis to insure they are operating within the manufacturer's specifications. They should also be tested anytime you suspect a problem may be present. One quick way to check the operation of two of the valves is to use a tracer compound and STEP gradient to monitor their operation. You can set up a method to perform this test as suggested below.

QUICK GRADIENT COMPOSITION TEST:

Bottle A = 100% DH20;
Bottle B = 0.1 % Acetone in DH20 (*Acetone is the tracer compound);

Flow Rate = 1.000 ml/min;
Column = No column. Install a restriction capillary in place of the column to obtain a backpressure of > 60 Bars;

Detection = 265nm (10 nm bandwidth) UV;

STEP Gradient Program:
    0 to 2.00 min, 0 % B
    2.01 min, 20% B
    4.01 min, 40% B
    6.01 min, 60% B
    8.01 min, 80% B
  10.01 min 100 % B
  12.01 min 20% B
  14.00 min 20% B

Note: If the delay volume (dwell volume) of your system is large, then you may want to adjust the time values shown to LARGER values (i.e. 2 minutes delays are used in this example, but 5 or even 10 minute delays between steps may be more appropriate if your system has > 1 ml dwell volume.

Running the above method should result in a signal trace which shows a step-wise rise to 12.00 minutes (as the acetone concentration increases). The edges of the "steps" should be sharp and the risers should also be close to vertical. The final step change which starts at 10.01 minutes shows a linear gradient change back down to the 20% B level. This line should not have any bumps or dips in it and should transition smoothly back down. The height of the baseline at this point should match the height seen between 2.01 and 4.00 minutes (same 20% B). The height of the proportional steps (e.g. 20, 40, 60, 80) should also be the same. You can use your CDS to measure these height values.

Another useful aspect to view is the S/N ratio at each step. Use your CDS to establish noise windows within each range (e.g. 2.50 to 3.5 minutes). This data is useful when comparing the performance of the pump at different intervals.

If you observe deviations in the height of the proportional steps or dips in the lines, these can be caused by leaking or sticking check valves as well as leaking or sticking gradient proportioning valves. *If you have a quaternary pump, be sure and test all four of the valves used (2x per test).

Lastly, the above example is a generalized method and may or may not be applicable to your specific HPLC pump. Be sure and customize a test method which takes into account the pressure ranges, flow rates, delay volume, mixing volume, and number of low pressure channels used in your pump.

External (ESTD) vs. Internal Standard (ISTD) Calibration in HPLC

Reliable quantitation of sample analytes using HPLC analysis requires accurate and reliable quantitation of a standard(s). For chromatography applications, we commonly use either an External Standard or an Internal Standard, as applicable, to insure reliable quantification of the sample.

• NOTE: A quick comment about calibration methods. Before you begin to create any calibration tables or analyze any standards/samples, please make sure that your current chromatography method follows good chromatography fundamentals. It must be selective for the sample type, retain the compound(s) with good K prime values, be reproducible and resolve apart all of the samples and possible impurities with near to perfectly symmetrical peak shapes. Your calibration results will only be as good as your original method. A poor quality method may not provide reliable results so be sure and spend as much time as possible developing the initial HPLC method to be as rugged and reliable as possible before starting any quantitation or calibration. *Poor quality method development is the number one reason for problems with quantitation.

Methods of Quantitation, Peak-height vs. Peak-area: Both types of response provide a measurement of the detector signal output. Proper and reproducible integration of the signal output is critical. Peak area is the most popular choice in chromatography, but peak height measurements can also be used if the peaks have near perfect symmetry (very rare, so peak area is far more reliable for integration). Whichever method you chose, you must use it consistently and document it well.

Definitions, External & Internal Standards: For most samples, there are two commonly used types of standards used. When known standards are run separately from the actual samples (in their own chromatogram) and their response is compared to that of the sample in another chromatogram, then we refer to this as an External Standard (ESTD). When the standard is added to the sample and analyzed at the same time we refer to this as an Internal Standard (ISTD). With an Internal Standard we are comparing the instrument's response to the sample to a reference standard with similar response characteristics, both run together.

External Standard (ESTD) Calibration Notes: The sample must fall within a range bracketed by the calibration solution. I suggest that you include a range which covers concentration values which are ~ 50% or more outside of the expected range. Dissolve the final calibration standards into the mobile phase (or a weaker solution) when preparing the injection vials from the stock solution. At least five (5) different concentration values should be used per order-of-magnitude (larger range = more stds). *Inject the same volume of solution (different concentration) for each calibration standard point ("level") onto the column. Do Not inject different volumes of solution from one std vial to create different concentrations. Plot peak response vs concentration. Ideally, you should have a linear response and the line will go through the origin (true zero intercept, ideally, though matrix effects/or the use on non std detectors such as the ELSD or CAD may require complex curve fits/formulas to describe the response). Once you have injected all of the standards, repeat the process again at least three more times (or use multiple injections) to determine overall reproducibility before constructing the final calibration table.

Internal Standard (ISTD) Calibration Notes: Internal standards are commonly used when many sample preparation steps are required before the sample can be injected onto the column. The internal standard may compensate for any losses during filtration or extraction. Selection of the Internal Standard is critical. Some of the characteristics of a good ISTD should include: It must be different than the sample, well resolved and must not elute where any sample peaks could be expected; It should not elute where any interfering matrix or other compounds could appear; It should have a similar linear response as the sample (Inject a fixed volume/concentration); Available in a high purity form from one or more commercial sources (certified method); Must be stable and not react with the sample or mobile phase solution. 

Add it to the samples before any extraction procedures. Base the amount of ISTD concentration such that it is between 1/3 and 1/2 of the expected concentration of the sample(s). The sample's target concentration range is a good value to use. *Because of these and other strict conditions, finding a suitable Internal Standard can take some time and testing.