HPLC Carry over and its Solution to prevent
Carryover Discription
Carryover is recognized as the presence of a small analyte peak that appears when a blank is injected following the injection of a sample that produces a large peak of the same analyte. When it occurs, peaks attributed to the previously analyzed sample may be observed in the subsequent chromatogram(s) which may co-elute or interfere with desired analytes. Shown below, the mass spectrum of a blank reveals the profile of a target analyte. It is one the most frustrating problems in HPLC.
Carryover usually amounts to a chemistry problem when coupled with certain sample injection flow path materials and hardware connections. What steps can we take to solve it?
Action Steps Overview
- Classify the carryover
- Replace the blank and vary the injection volume
- Rule out the chromatography column
- Check flow path fittings
- Check autosampler rinse phase solvent(s)
- Enable autosampler rinse mechanism
- Change hardware
- Addressing sample-specific carryover
Take Action
1. Classify the carryover
ii. Constant carryover – Not true carryover. A small peak is always present with all samples and blanks and does not diminish with each blank run. This is caused by some source of contamination. If the contaminant peak seems to increase or decrease with increasing and decreasing blank injection volumes, it is likely that the blank is contaminated. If not, the source of contamination is elsewhere in the system.
iii. Consider the importance of a ‘null-injection’ run. Shimadzu autosamplers offer the unique ability to perform a null-injection, which starts the chromatography run without injecting sample and without rotating the injection valve or high pressure valve of the autosampler. This simple test can help to determine whether the source of the problem is the injection event itself. A null run that produces no offending peak must indicate that the auto-sampler injection event is the source of the issue. It does not prove the source of the carryover within the autosampler, but it does narrow the problem down to the high pressure valve (HPV), its connections, and the outlet tubing connected to the column. Because Shimadzu autosamplers are needle-in-flow path design, this experiment rules out the sample loop and needle because eluent is always flowing through the loop and needle unless the valve is rotating or sample is being aspirated from a vial.
2. Replace the blank and vary the injection volume
3. Rule out the chromatography column
4. Check flow path fittings
5. Check autosampler rinse phase solvent(s)
ii. Use fresh rinse solvent - Replace the rinse phase solvents with a fresh batch and replace the rinse phase reservoirs with clean ones. After the rinse solvent is replaced, cycle the autosampler through the purge or rinse cycle several times to ensure that all traces of the previous rinse phase solvents have been removed.
6. Enable autosampler rinse mechanism
7. Change hardware
ii. Sometimes the sample adsorbs on the sample loop because of the characteristics of sample and injection solvent. Replacing the loop with one of different composition can help solve this problem. Injection loops are available in stainless steel and PEEK. A combination of a new loop material and a different injection solvent should eliminate sample adsorption on the loop.
iii. The internal components of the HPV will become worn over time as a normal consequence of doing business. A worn rotor can be the source of carryover, but it is also usually associated with impairing the quality of chromatography peaks. Consider repairing or replacing the HPV.
8. Addressing sample-specific Carryover
Reducing Carryover Using Autosampler Rinsing
As an overall rationale for addressing carryover with autosampler rinsing, use external rinsing first as it is quick and easy. If a more aggressive approach is desired, try adding internal rinsing.
External Rinse with Autosamplers
This cleans the outer surface of the needle by immersing it and/or actively rinsing it in a rinse port before or after the sample is drawn. There are two rinse ports (immersion and flow). In simplest terms, the immersion rinse port allows you to dip the needle into a rinse solvent (R0). Commonly, R0 is mobile phase or something chemically stronger. A multi-rinse option (equipped as standard on the SIL-40C X3) allows you to use an active flow rinse port to clean the outer surface of the needle with a different combination of rinse solvents (R1 and R2). The R1 and R2 could set up a rinsing sequence of strong solvent (R1) followed by a weaker solvent (R2). The multi-rinse option may also use an active rinse solvent (R3), which controls cleaning with an active diaphragm pump.
Internal Rinse with Autosamplers
This function can rinse the inner surface of the needle and sample loop, and the injection port. Use of the multi-rinse kit enables rinse with up to 3 types of solution (R0, R1, and R2). The strong/weak rationale should be used when setting choosing the chemistry of the rinse phase solvents. The following table summarizes available rinse options.