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Chromatography, electrophoresis, mass spectrometry and osmometry

15 min

  • Predict elution order from how compounds interact with the stationary phase
  • Predict protein migration from buffer pH relative to the isoelectric point
  • Check the internal-standard response before releasing an LC-MS result
  • Choose an osmometry principle that detects volatile solutes

Read the full reference

Try first

Try first

A drug and its much more polar metabolite are separated on a reversed-phase C18 column. Which one elutes first?

The next section explains it.

Right. The next section explains why.

The next section explains it.

The next section explains it.

Get the idea

Chromatography separates by interaction

Each compound divides its time between a moving mobile phase and a fixed stationary phase. The more a compound interacts with the stationary phase, the later it elutes.1,2

  • Reversed phase. A nonpolar bonded phase, commonly C18, holds nonpolar compounds longest. Polar compounds elute first.
  • Size exclusion. Large molecules cannot enter the pores of the packing, so they elute first.

A retention time or a thin-layer Rf supports identity only within one defined system and against standards run under the same conditions. Two compounds can coelute. A second solvent system, a color reaction or mass spectrometry supplies the confirming evidence.1,2

Electrophoresis follows net charge

Buffer pH sets the sign of a protein's charge relative to its isoelectric point (pI).1,2

  1. Above its pI, a protein carries a net negative charge and moves toward the anode.
  2. Below its pI, it carries a net positive charge and moves toward the cathode.
  3. At its pI, net movement approaches zero.

Serum protein electrophoresis commonly runs at pH 8.6, above the pI of most serum proteins, so they move toward the anode.1 Electroendosmosis moves buffer toward the cathode and can carry weakly charged proteins with it.2

The internal standard in LC-MS

Quantitative liquid chromatography-mass spectrometry (LC-MS) adds a stable-isotope-labeled internal standard to each specimen early in preparation. The result comes from the ratio of analyte response to internal-standard response. That ratio corrects losses in extraction, injection and ionization to the extent the two compounds behave alike.3

The internal-standard response has its own acceptance limits. A sharp drop in one specimen points to ion suppression or a preparation failure that the ratio may not fully correct. Re-extract or dilute that specimen and run it again before any result is released.2,3

Freezing point or vapor pressure

Freezing-point depression counts every dissolved particle. Each mOsm/kg lowers the freezing point by 0.00186 °C.1 Ethanol is volatile and enters the vapor phase, so it adds little to a vapor-pressure (dew-point) result. When an osmolal gap is calculated to look for a volatile solute such as ethanol, measure osmolality by freezing point, and keep the specimen capped so the ethanol does not evaporate.1,2

References
  1. Bishop ML, Fody EP, Van Siclen C, Mistler JM, Moy M. Clinical Chemistry: Principles, Techniques, and Correlations. 9th ed. Jones & Bartlett Learning; 2023.
  2. Rifai N, Chiu RWK, Young I, Burnham CAD, Wittwer CT, eds. Tietz Textbook of Laboratory Medicine. 7th ed. Elsevier; 2023.
  3. Clinical and Laboratory Standards Institute. Liquid Chromatography-Mass Spectrometry Methods. 3rd ed. CLSI guideline C62. Clinical and Laboratory Standards Institute; 2026. Accessed September 26, 2026. https://clsi.org/shop/standards/c62/

Watch one

The emergency department sends serum for measured osmolality so that an osmolal gap can be calculated. The patient is suspected of drinking a large amount of alcohol. The tube arrives capped and full. Your laboratory has a vapor-pressure osmometer on the main line and a freezing-point osmometer in the stat area. Which instrument do you use, and how do you handle the tube?

  1. Name the question: the gap is calculated to find unmeasured solutes, and ethanol is one of them.

    The purpose of the test decides which measurement principle fits it.

  2. Rule out the vapor-pressure osmometer: it would leave most of ethanol's share out of the measured osmolality and shrink the gap.

    Ethanol enters the vapor phase and adds little to a dew-point reading.

  3. Choose the freezing-point osmometer: it counts ethanol with every other dissolved particle.

    Freezing-point depression responds to particle number, whatever the particle.

  4. Keep the tube capped until the sample is taken, and analyze it promptly.

    Ethanol lost from an open tube is lost from the result too.

Measure osmolality on the freezing-point osmometer, with the tube kept capped until analysis.

Your turn

Problem 1 of 3

A protein with an isoelectric point of 5.0 is placed in a buffer at pH 8.6 and the field is switched on. Which way does it move?

Movement toward the cathode needs a net positive charge, which a protein has below its isoelectric point. At pH 8.6 this protein is well above its pI.

Reversed migration above the isoelectric point

Above its isoelectric point a protein carries a net negative charge and migrates toward the anode. Below it, the net charge is positive and the protein moves toward the cathode. Reversing the direction misassigns bands. Electroendosmosis can also carry weakly charged proteins toward the cathode.

A protein stops moving only near its isoelectric point. At pH 8.6 it is 3.6 pH units above its pI and carries a clear net charge.

Above its isoelectric point the protein carries a net negative charge, so it moves toward the positive electrode, the anode.

Hint
  1. Compare the buffer pH with the protein's isoelectric point.
  2. The buffer pH is higher than the isoelectric point. Work out the sign of the net charge.
  3. A charged particle moves toward the electrode of opposite charge.

Review Electrophoretic separation

Problem 2 of 3

A thin-layer chromatography drug screen on urine shows a spot with the same Rf as the amphetamine standard on the same plate. What does the spot support?

Unrelated compounds can migrate to the same position in one system. A color reaction, a second solvent system or mass spectrometry supplies the confirming evidence.

Running the standard on the same plate makes the comparison fair. It cannot rule out another compound with the same Rf.

Treated matching retention as definitive identification

Two compounds can share a retention time or spot position under one set of conditions. Reporting identity from retention alone can name the wrong drug. A second solvent system, a color reaction, or mass spectrometry supplies the confirming evidence.

Thin-layer chromatography does separate and detect drugs. Its spot position gives a presumptive identification.

Hint
  1. Ask whether any other compound could travel to the same position on this plate.
  2. Retention and Rf describe behavior in one solvent system.

Review Chromatographic separation

Problem 3 of 3

A mixture of IgM (about 900 kDa), albumin (about 66 kDa) and β2-microglobulin (about 12 kDa) is run on a size-exclusion column. Which protein elutes first?

The smallest protein enters the most pores and travels the longest path through the packing. It elutes last.

Albumin enters some of the pores. It elutes after IgM and before β2-microglobulin.

IgM is too large to enter the pores, so it passes around the packing and leaves the column first.

Size exclusion separates by hydrodynamic size, and these three proteins differ widely in size.

Review Chromatographic separation

Use it

  • A night batch of 40 whole-blood tacrolimus specimens runs by LC-MS with tandem detection.
  • Calibrators and both control levels are within limits. Every other specimen's internal standard is within limits.
  • The procedure accepts an internal-standard peak area from 50% to 150% of the mean area of the run's calibrators.
  • One transplant patient's specimen (MRN 0072915) gives the results below.
TestResultPreviousReference intervalFlag
Tacrolimus12.9 ng/mL7.8 ng/mLSep 24, 06:05
Internal-standard area18 %97 %Sep 24, 06:0550–150 %Low

Specimen: Not measured on whole blood. Whole blood, EDTA. Collected 05:50 before the morning dose.

Decision 1 of 3

What does the internal-standard area tell you?

The ratio corrects only as far as the analyte and the internal standard behave alike. A response this far outside its limits shows something that may have affected them differently.

Ignored a low internal-standard response

The analyte-to-internal-standard ratio corrects extraction, injection, and ionization only as far as the two compounds behave alike. A sharp drop in internal-standard response signals ion suppression or a preparation failure that the ratio may not fully correct, so the specimen is re-extracted or diluted and rerun before the result is released.

The calibrators, both controls and every other specimen's internal standard are within limits. The problem sits with this one specimen.

The internal standard is added in the same amount to every specimen, so its area does not depend on the patient's drug level.

One specimen with an internal-standard area of 18% in an otherwise acceptable run points to its own matrix or preparation. The ratio may not have corrected it.

Review Sources and analyzers

Decision 2 of 3

What do you do with the tacrolimus result?

A fresh extraction, diluted if the procedure allows, tests whether the internal standard now recovers. The result waits until it does.

The comment would not make the number reliable. The 12.9 ng/mL could reach the chart as the patient's level.

Nothing in the calibrators, controls or other specimens shows a run problem. The action belongs with this one specimen.

Review Sources and analyzers

Decision 3 of 3

The re-extracted specimen gives an internal-standard area of 96% and tacrolimus 7.6 ng/mL. The peak sits at the expected retention time, and its transition ratio is within limits. What do you report?

The first run failed its internal-standard check, so its result has no place in the report. Averaging carries its error into the number.

The rerun meets every acceptance check the procedure names: internal-standard area, retention time and transition ratio. Its result is the one to report.

Only a result that met its checks is reported. The first run's value describes a failed preparation.

Review Sources and analyzers

The clue that settles this case is the internal-standard area of 18% in a run where everything else passed. That points to this specimen alone. A fresh extraction with an acceptable internal standard gives the result the laboratory reports.

Keep

Sources checked