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.

Two Common HPLC Problems and their Causes (Sudden changes to either the HPLC Backpressure or Peak Shape)

   Let's take a quick look at two different problems which you may encounter when operating an HPLC system. We start with the basic observation and then look at the most likely causes so we can begin the troubleshooting process and repair the problem. An automated HPLC system's flow path typically consists of: The Solvent Pickup Filters (in the mobile phase reservoirs); The Pump(s); AutoSampler; AutoInjector; Column and one or more Detectors.*You should have a good understanding of this flow path before you proceed to diagnose the problem(s).

 *A gradual increase of pressure for the same method over time is often due to column fouling or a dirty inlet frit (e.g. PTFE frit). This article specifically focuses on the causes of a sudden change, not a slow change over time.

   Sudden System Back Pressure Changes: We will assume that you have been running the same method for some time or at least several times without a problem and then suddenly notice that the back pressure has changed from what is normally seen. The problem must lie within the flow path of the system.

   Excessive High Pressure: Typical reasons for this are:

1.      A fouled or plugged column;
2.      Wrong flow rate (higher than normal);
3.      Inlet frit/filter plugged or restricted;
4.      Plugged line;
5.      Wrong mobile phase composition.

   Large Drop in Pressure: Typical reasons for this are:

1.      A leak at a fitting, column or line (Number one reason);
2.      Wrong flow rate (lower than normal);
3.      Wrong mobile phase composition. 

• Start by checking the method parameters to insure that they have not changed (i.e. flow rate, mobile phase composition). Check for leaks or plugs. If the column is suspect, replace it with a zero dead volume union (ZDU) and restrictor and flush the system. Replace the column with a new one or wash the current column according to the column manufacturer's guidelines.

   Sudden Peak Shape Changes: We will again assume that you have been running the same method for some time or at least several times without a problem and then suddenly notice that the peak shape of one or all of the peaks has changed from what is normally seen. *The key thing to keep in mind is that the change occurs all of a sudden, not because of poor initial method development.

   Typical reasons for this are:

1.      Tailing or Split Peaks: Sample overload, change in flow rate, mobile phase composition (e.g. composition or pH), void formation, dirty frit, injection solvent too strong or a fouled column.
2.      Fronting: Commonly seen when overloading sample on column.
3.      Ghost Peaks: Usually due to a contaminated mobile phase, contaminated sample vial or contaminated injector (e.g. rotor seal).
4.      Broad Peaks: Large sample injection volumes or extra column volume (bad connections with the system or tubing) are usually to blame. Try reducing the injection volume by a factor of 10 and see if the problem goes away. You may also want to wash the column as it may be fouled with sample.

   These are just two common problems we see when using HPLC systems. Note that a dirty or fouled column can cause many of these problems so take care of your columns and wash and test them regularly to insure they are in compliance. There are many other commonly seen problems besides these. If you would like to see a specific problem featured on this blog, then please send me a request.