Choosing an HPLC Column for a Regulated Method: USP L-Designations, Equivalency and Revalidation

Leave a Comment / By / October 18, 2026

A USP L-designation names a packing type, not a column: many dissimilar C18 columns are all L1. To replace the column in a USP monograph method, keep the same bonded substituent, rank candidates by their USP–PQRI similarity value F (≤ 3 suggests equivalence), stay within the ⟨621⟩ adjustments and pass system suitability. Other changes need revalidation.1,2

What does a USP L-designation specify, and what does it leave open?

USP General Chapter ⟨621⟩ states that the type of packing needed for an analysis is indicated by the “L” designation in the individual monograph, and that typical column dimensions are given there too. Monographs do not name columns, which avoids the appearance of endorsing a vendor’s product and allows for natural changes in the marketplace.1

The designation is therefore a category. Dolan and Snyder describe the L classification as oversimplified: all C18 columns are L1, different L1 columns can give very different chromatograms, and a C18 and a phenyl column from one manufacturer may be more similar than two C18 columns from different manufacturers.3 The phase families and their packing codes are compared in how stationary-phase chemistry changes selectivity. For basic analytes, the silica and endcapping behind an L1 label often decide the peak shape, as explained in Type A vs Type B silica and endcapping.

Table 1 sets what the pharmacopeia fixes against what it leaves to the laboratory.

Table 1. What a USP monograph fixes about the column and what it leaves open, from the Stage 4 harmonized text of ⟨621⟩ and the USP Chromatographic Columns database.1,4
Column property What USP fixes Where it is decided
Bonded substituent (octadecyl for L1) Set by the L-designation; no change of its identity, so no C18 to C8 The monograph1
Support, surface modification and extent of chemical modification (endcapping, carbon load) Must be similar to the original; the L-code does not define them The laboratory’s comparison of candidates1
Length, internal diameter and particle size Typical values in the monograph, adjustable within ⟨621⟩ limits ⟨621⟩ adjustment rules1
Particle type (totally or superficially porous) A change to superficially porous particles is allowed if the stationary-phase requirements are met; other length and particle-size combinations then need the plate number within −25% to +50% and equivalent selectivity and elution order of the specified impurities ⟨621⟩ adjustment rules1
Brand Not named in the monograph The USP database lists the column used in validation4
Selectivity Not described by any L-code Hydrophobic-subtraction parameters, then experiment3
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Where do you find the column used to validate a monograph method?

USP’s Chromatographic Columns database is an online compilation of the columns used to validate the chromatographic procedures in USP–NF, Pharmacopeial Forum, Revision Bulletins and Interim Revision Announcements, covering revisions since January 1980. For each column it gives the L, G or S designation, the type of test, brand names, manufacturers and alternative columns where that information is available. It is free after a one-time registration.4

Read its disclaimer before relying on it. Brand names are listed to show market availability; a listing does not imply approval or endorsement, the list is not comprehensive, it is compiled from supplier information whose accuracy cannot be guaranteed, and USP has no particular knowledge of a listed product’s continued suitability.4 A listed brand is a candidate, not proof. As LabVeda practice, start with the validation column where it is still sold, because the monograph’s chromatographic conditions were validated during its elaboration.1

Figure 1 maps the chain from the monograph to a column you can use.

Map of the route from a USP monograph to a replacement column. Band A, five tiles joined by arrows: 1, Monograph (a page with an L1 chip): names the L-code, typical dimensions and system suitability criteria; no brand. 2, USP column database (a listing with one row highlighted): lists the column used in validation, brands and alternatives; a listing is not an endorsement. 3, Same substituent (C18 labelled L1 with a tick, C8 labelled L7 with a cross): no change of substituent identity; a C18 stays L1; a C8 (L7) is a modification. 4, Rank by Fs (schematic bars with an Fs = 3 line): USP–PQRI comparison at pH 2.8 or 7.0; Fs ≤ 3 suggests equivalence. 5, USP ⟨621⟩ and system suitability (an L/dp change bar from −25% to +50% above an original and a replacement trace with the same peaks): L/dp within −25% to +50%; pass system suitability on the real sample. Band B, What an L-code leaves open: support, surface modification, extent of modification must be similar to the original under USP ⟨621⟩; brand not named in the monograph; selectivity not described by any L-code.
Figure 1. From monograph to a usable column. The L-designation fixes the substituent; the USP database names the column used in validation, the USP–PQRI comparison ranks candidates by Fs, and ⟨621⟩ plus system suitability decide whether the change is an adjustment.1,4,2 The lower band lists what an L-code leaves open.1,3 Fs ≤ 3 is a guideline for reversed-phase alkylsilica columns, not an acceptance criterion. Schematic icons, not data.

How do you judge whether a replacement column is equivalent?

The USP–PQRI procedure rests on almost 3,000 retention-time measurements for 150 test compounds on 100 alkylsilica columns, which showed that five column properties account for selectivity: hydrophobicity (H), steric interaction (S*), hydrogen-bond acidity (A), hydrogen-bond basicity (B) and ion-exchange capacity (C). Because C depends on mobile-phase pH, the program compares columns at pH 2.8 or 7.0, whichever is closer to the method, and asks whether acids or bases are present.2

The similarity of two columns is reported as F (written Fs in the literature), a weighted distance in the five-parameter space:5

Fs = {[12.5(H2 − H1)]2 + [100(S*2 − S*1)]2 + [30(A2 − A1)]2 + [143(B2 − B1)]2 + [83(C2 − C1)]2}1/2

Subscripts 1 and 2 denote the original and candidate columns. The weights are adjustable but usually taken as shown; the web tools do the calculation.5 Use the PQRI database rather than the smaller USP C18 set on the same site, which Dolan and Snyder describe as less fully validated.3 Table 2 gives the published readings of Fs.

Table 2. Reading Fs when looking for an equivalent reversed-phase column. Guidelines, not limits, as stated by Dolan and Snyder; the USP–PQRI page gives them as likelihoods.2,3
Fs Reading Source
≤ 3 Very likely to give an equivalent, acceptable separation for any sample USP–PQRI2
3 to ≈10 Likely equivalent, especially for samples with few solutes and Rs well above 2 Dolan and Snyder3
Smallest available The candidate most likely to give a similar, adequate separation USP–PQRI2
Any value Confirm with the same sample under the same conditions on both columns Dolan and Snyder3

Three cautions apply. The model is less reliable for phenyl and cyano columns, so replace those with the same column type and a small Fs.3 The column nearest by Fs can belong to a different phase type, which Stoll notes creates challenges in a regulated environment;5 under ⟨621⟩ the substituent cannot change, so such a column is not an adjustment.1 And a large Fs serves the opposite job, a second column with different selectivity, covered in choosing an orthogonal HPLC column for impurity profiling.

Which column changes count as adjustments under USP ⟨621⟩?

The Stage 4 harmonized text of USP General Chapter ⟨621⟩ lists how far each condition may be adjusted without fundamentally modifying the procedure; changes other than those require revalidation. Multiple adjustments can have a cumulative effect and must be evaluated, adjustments are made from the pharmacopeial procedure, and once a procedure has been adjusted no further adjustments are allowed without revalidation. Additional verification tests may be required, assessing the performance characteristics the change could affect, and system suitability must be met.1

For the column, the isocratic and gradient rules are parallel, with the one difference noted in the third item:1

  • No change of the identity of the substituent; the support, surface modification and extent of chemical modification must be similar.
  • Length and particle size may change if L/dp stays within −25% to +50% of the prescribed ratio; internal diameter may change if particle size and length do not.
  • A change from totally porous to superficially porous particles may instead keep the plate number N (isocratic) or (tR/Wh)2 (gradient) within −25% to +50%, provided system suitability passes and selectivity and elution order of the specified impurities are shown to be equivalent.
  • Flow rate is recalculated for internal diameter and particle size.
  • A guard column may be no longer than 15% of the analytical column, of the same or smaller internal diameter, with the same packing and bonded phase.

Table 3 applies these rules to five candidates for an assumed monograph that specifies an L1 column of 250 × 4.6 mm, 5 µm (L/dp = 250/5 = 50) at 1.0 mL/min. The flow column uses the ⟨621⟩ equation:1

F2 = F1 × [(dc22 × dp1)/(dc12 × dp2)]

Here dc is the internal diameter and dp the particle size of the original (1) and new (2) columns. For candidate D, 1.0 × (3.02 × 5)/(4.62 × 2.7) = 45/57.13 = 0.79 mL/min.

Table 3. Worked check of five candidate columns against an assumed L1 monograph column (250 × 4.6 mm, 5 µm, 1.0 mL/min), applying the ⟨621⟩ stationary-phase, L/dp and flow rules. LabVeda calculations; candidates are hypothetical.1
Candidate Substituent L/dp (change) Flow ⟨621⟩ status
A. C18, 250 × 4.6 mm, 5 µm, another brand C18 (L1) 50.0 (0%) 1.0 mL/min Adjustment if the other stationary-phase characteristics are similar; rank by Fs
B. C18, 150 × 4.6 mm, 3 µm C18 (L1) 50.0 (0%) 1.67 mL/min Adjustment
C. C18, 100 × 4.6 mm, 3 µm C18 (L1) 33.3 (−33.3%) 1.67 mL/min Outside the L/dp window: revalidation
D. C18, 100 × 3.0 mm, 2.7 µm superficially porous C18 (L1) 37.0 (−25.9%) 0.79 mL/min Outside the L/dp window; acceptable only through the plate-number route with equivalent selectivity and elution order
E. C8, 250 × 4.6 mm, 5 µm C8 (L7) 50.0 (0%) 1.0 mL/min Substituent changes: revalidation, whatever the dimensions

Candidate C shows why the arithmetic matters: with 3 µm particles, a 100 mm column falls a third below the prescribed ratio. The flow, gradient-time and injection-volume arithmetic for dimension changes, with a gradient example, is worked through in how to scale an HPLC method to new column dimensions. A move to another mode, such as from reversed phase to HILIC, replaces the substituent entirely, and the reasons to make that move are covered in when to switch from reversed-phase HPLC to HILIC.

Figure 2 separates the column changes ⟨621⟩ treats as adjustments from those that need revalidation.

Comparison of column changes under USP ⟨621⟩ in three bands. Band A, listed in USP ⟨621⟩: adjust, then verify: same substituent with support, surface modification and extent of chemical modification similar (a C18 particle); length and particle size changed with L/dp within −25% to +50% (a long and a shorter column); internal diameter changed when particle size and length are not, flow recalculated (a wider and a narrower column); totally to superficially porous particles with N, or (tR/Wh)² for gradients, within −25% to +50% and equivalent selectivity and elution order of the specified impurities (a porous particle and a core-shell particle); guard column no longer than 15% of the column, same or smaller internal diameter, same packing and bonded phase. Band B, not an adjustment: revalidate: substituent changed, for example C18 to C8 (C18 and C8 particles); L/dp outside the window (a short column with a cross); a further adjustment of a procedure already adjusted (a page with a loop arrow); any change not listed in USP ⟨621⟩ (a page with a question mark). Band C: an L/dp change bar from −25% to +50% with the Table 3 candidates marked: A (250 mm, 5 µm) and B (150 mm, 3 µm) at 0% inside, D (100 mm, 2.7 µm) at −25.9% and C (100 mm, 3 µm) at −33.3% outside; D can still qualify through N, and E (C8) needs revalidation whatever its dimensions.
Figure 2. Adjustment or revalidation? Column changes that the Stage 4 text of ⟨621⟩ treats as adjustments, verified by system suitability, against those that require revalidation.1 The window bar marks the Table 3 candidates for an assumed 250 × 4.6 mm, 5 µm L1 column: A (250 mm, 5 µm, another brand) and B (150 mm, 3 µm) at 0%, D (100 mm, 2.7 µm, superficially porous) at −25.9% and C (100 mm, 3 µm) at −33.3%; D can still qualify through the plate-number route, and E (C8) needs revalidation whatever its dimensions. LabVeda calculations; schematic, not data.

These rules have a limited scope. ⟨621⟩ applies only to monograph methods published in the USP, not to methods developed and validated in a laboratory, taken from the literature or inherited; its rules can be adopted in a laboratory SOP, but they are then the laboratory’s guidelines.6 Ph. Eur. 2.2.46 incorporates the same harmonized text, signed off by the Pharmacopoeial Discussion Group on 28 September 2021 and implemented from 1 January 2023.7 For validated or compendial methods, the applicable procedure and regulatory framework take precedence over the general rules of thumb given here.

When is verification enough, and when does a column change need revalidation?

For a USP method that is not modified, US GMP regulations accept a statement of the method and its reference, but the suitability of every testing method must be verified under actual conditions of use. Any modification of an established method needs complete records, including the reason and data showing that the modified method is at least as accurate and reliable as the established one.8

ICH Q14 defines revalidation as a demonstration that a procedure is still fit for its intended purpose after a change, covering all or a subset of the performance characteristics. For a change within the same analytical procedure principle, its examples call for partial or full revalidation of the characteristics affected and, as appropriate, comparative analysis of representative samples and reference materials. In its illustrative chiral HPLC example, the column chemistry is an established condition assessed as low risk, while column length and particle size are not, because the system suitability test controls them; reporting categories differ by region.9

As LabVeda practice, treat a column change within the ⟨621⟩ adjustments, with system suitability passed and the change documented, as verification. A change outside them is a modification, which ⟨621⟩ says requires revalidation; keep the records of its reason and supporting data that the regulation asks for.1,8 Table 4 sets out the decision path in order.

Table 4. Choosing a replacement column for a USP monograph method. Steps are sequential; the ⟨621⟩ conditions apply to monograph methods only.1,2,6
Step What to do Move on when
1. Identify Read the L-code, dimensions and system suitability criteria; look up the validation column in the USP database A candidate list exists4
2. Same substituent? Confirm each candidate’s bonded phase and L-code match the monograph Yes; if not, the change needs revalidation1
3. Rank by Fs Compare in the PQRI database at the method pH, with acids and bases flagged Fs ≤ 3, or a larger value only for an easy separation2
4. Check ⟨621⟩ L/dp (or N for superficially porous particles) and the flow equation Within the limits; if not, the change needs revalidation1
5. Verify Run system suitability and the same sample on both columns; assess affected characteristics; document Criteria met and records complete; if not, try the next candidate or revalidate1,3

Figure 3 follows Table 4 step by step.

Five-step workflow matching Table 4, joined by arrows: 1, Identify (a monograph page with an L1 chip): read the L-code, dimensions and system suitability criteria and look up the validation column in the USP database; move on when a candidate list exists. 2, Same substituent? (C18 labelled L1 with a tick, C8 labelled L7 with a cross): confirm each candidate's bonded phase and L-code match the monograph. 3, Rank by Fs (schematic bars with an Fs = 3 line): compare in the PQRI database at the method pH with acids and bases flagged; Fs ≤ 3, or a larger value only for an easy separation. 4, Check USP ⟨621⟩, monograph methods only (an L/dp change bar from −25% to +50% with a tick inside and a cross outside): L/dp, or N for superficially porous particles, and the flow equation. 5, Verify (the same three peaks in the same order on the original and the replacement column, with a tick): run system suitability and the same sample on both columns, assess affected characteristics, document. Exits: No at step 2 and Outside the limits at step 4 lead to a Revalidate box; at step 5, Criteria met leads to Verified and documented, Not met loops back to step 3 for the next candidate, or revalidate.
Figure 3. Choosing a replacement column for a monograph method. The figure follows Table 4 row for row; steps are sequential. Identify the monograph’s column requirements and the validation column, keep the substituent, rank candidates by Fs, check the ⟨621⟩ limits, then verify with system suitability and the same sample on both columns and document the change.1,4,2 The exits are drawn: a changed substituent or a change outside the limits leads to revalidation; a candidate that fails verification sends you back to step 3 for the next one, or to revalidation. The ⟨621⟩ conditions apply to monograph methods only.6 Schematic icons and traces, not data.

How can a new method be written so its column can be replaced later?

Plan for the replacement during development. Stoll describes the common practice of identifying, late in development, a column with very similar selectivity that can serve as a drop-in replacement if the manufacturer stops making the primary column or cannot make it reproducibly; the database produces a short list to test experimentally.5 ICH Q14 asks that the outcome of robustness work be reflected in the analytical procedure control strategy, and the system suitability test is designed to detect unacceptable performance.9 Column choice is one step of how to develop an HPLC method. As LabVeda practice, record the tested alternative column and its Fs with the method, so a later substitution starts from data rather than a catalog.

Frequently asked questions

Is any C18 column acceptable when a monograph specifies L1?

Not automatically. L1 fixes the octadecyl substituent, but ⟨621⟩ also requires the support, surface modification and extent of chemical modification to be similar, and system suitability must pass. Different L1 columns can give very different chromatograms, so rank candidates by their USP–PQRI Fs against the validation column and confirm on the real sample before routine use.1,3

What does Fs ≤ 3 mean in the USP–PQRI comparison?

It is the guideline below which a candidate is very likely to give an equivalent, acceptable separation for any sample. For easy separations, with resolution well above 2, a larger value may still work, and the smallest available value is the best bet. It is a guideline for reversed-phase alkylsilica columns, not an acceptance criterion, and the match still has to be confirmed with the same sample and conditions on both columns.2,3

Can a C8 (L7) column replace a C18 (L1) column under ⟨621⟩?

No. The harmonized text allows no change in the identity of the substituent and gives C18 to C8 as its example. Even if a C8 column gave an acceptable chromatogram, using it is a modification of the procedure, which needs revalidation of the affected performance characteristics and complete records of the change and its justification.1,8

Can a superficially porous column be used in a USP monograph method?

Yes, with conditions. ⟨621⟩ allows a change from totally porous to superficially porous particles when the stationary-phase requirements are met. Because these columns give more plates than their particle size suggests, the length and particle size may be chosen by keeping the plate number within −25% to +50% instead of L/dp, provided system suitability passes and the selectivity and elution order of the specified impurities are shown to be equivalent.1,6

Do the ⟨621⟩ column adjustments apply to an in-house validated method?

No. They apply only to monograph methods published in the USP. Methods developed and validated in your laboratory, taken from the literature or inherited fall under their own validation and change control. Many laboratories adopt similar limits in an SOP, but those limits are then the laboratory’s own rules and must be justified by its own data and procedures.6

Does a column change in an approved method need regulatory notification?

That depends on the established conditions in the application and on regional rules. ICH Q14 treats changes through risk assessment and bridging studies; in its illustrative example the column chemistry is an established condition assessed as low risk, while column length and particle size are controlled by system suitability and are not established conditions. Check your own registration before changing the column.9

The takeaway

An L-designation narrows the choice to a packing type; it does not choose the column. Start from the validation column in the USP database, keep the substituent, rank candidates by USP–PQRI Fs, check the ⟨621⟩ limits and prove the replacement with system suitability and the real sample. Anything outside those limits is a modification that needs revalidation.1,4,2

References

  1. United States Pharmacopeial Convention. USP General Chapter ⟨621⟩ Chromatography, Stage 4 harmonized text (official 1 December 2022). https://www.usp.org/sites/default/files/usp/document/harmonization/gen-chapter/harmonization-november-2021-m99380.pdf (accessed 2026-10-17) — L designation indicates the packing; monographs give typical dimensions and do not name columns; adjustment of chromatographic conditions (changes other than those listed require revalidation, cumulative adjustments, no further adjustment without revalidation, verification tests); stationary-phase rule (no change of substituent identity, e.g. C18 to C8); L/dp −25% to +50%; TPP to SPP by N or (tR/Wh)²; flow equation; guard column.
  2. United States Pharmacopeia. USP–PQRI procedure for choosing a replacement reversed-phase column. https://usp.org/node/12526 (accessed 2026-10-17) — five column parameters from 100 alkylsilica columns; C depends on pH (2.8 or 7.0); acids and bases boxes; F ≤ 3 very likely equivalent; larger F for easy separations; smallest F best.
  3. Dolan JW, Snyder LR. The hydrophobic-subtraction model for reversed-phase liquid chromatography: a reprise. LCGC North America. 2016;34(9):730–741. https://www.chromatographyonline.com/view/hydrophobic-subtraction-model-reversed-phase-liquid-chromatography-reprise — L classification oversimplified (all C18 are L1; C18 and phenyl from one maker can be more similar than two C18s); Fs guidelines (≤ 3; 3 to ≈10); PQRI database recommended; lower reliability for phenyl and cyano; confirm matches experimentally.
  4. United States Pharmacopeia. Chromatographic Columns database. https://www.usp.org/resources/chromatographic-columns (accessed 2026-10-17) — columns used to validate USP–NF procedures since 1980; designations, brands, manufacturers, alternative columns; listing is not endorsement and is not comprehensive.
  5. Stoll DR. Selectivity in reversed-phase liquid chromatography: 20 years of the hydrophobic subtraction model. LCGC Europe. 2020;33(4). https://www.chromatographyonline.com/view/selectivity-reversed-phase-liquid-chromatography-20-years-hydrophobic-subtraction-model-0 — Fs as a weighted distance with weights 12.5, 100, 30, 143, 83; the most similar column can belong to a different phase type, a challenge in regulated work; identifying a drop-in replacement column late in development.
  6. Dolan JW. Method adjustment the USP way. LCGC Asia Pacific. 2017;20(2). https://www.chromatographyonline.com/view/method-adjustment-usp-way — ⟨621⟩ applies only to USP monograph methods, not in-house, literature or inherited methods; SOP use; superficially porous particles and the plate-number allowance.
  7. European Directorate for the Quality of Medicines & HealthCare. General chapter 2.2.46. Chromatographic separation techniques now published in Ph. Eur. 11th Edition. https://www.edqm.eu/en/-/general-chapter-2.2.46.-chromatographic-separation-techniques-now-published-in-ph.-eur.-11th-edition (accessed 2026-10-17) — Ph. Eur. 2.2.46 harmonized with USP ⟨621⟩; PDG sign-off 28 September 2021; implementation 1 January 2023.
  8. US Food and Drug Administration. 21 CFR 211.194 Laboratory records. https://www.ecfr.gov/current/title-21/chapter-I/subchapter-C/part-211/subpart-J/section-211.194 (accessed 2026-10-17) — (a)(2) unmodified compendial methods referenced, suitability verified under actual conditions of use; (b) records of any modification with reason and data.
  9. International Council for Harmonisation. ICH Q14 Analytical Procedure Development. Step 4, 1 November 2023. https://database.ich.org/sites/default/files/ICH_Q14_Guideline_2023_1116.pdf — revalidation definition; Table 2 change within the same principle; robustness reflected in the control strategy; SST purpose; Annex A example (column chemistry an established condition; length and particle size controlled by SST).

Further reading

  • Snyder LR, Kirkland JJ, Dolan JW. Introduction to Modern Liquid Chromatography. 3rd ed. Wiley.
  • Neue UD. HPLC Columns: Theory, Technology, and Practice. Wiley-VCH.
  • Dong MW. Modern HPLC for Practicing Scientists. Wiley.

Reviewed against cited sources. Column-adjustment statements follow the Stage 4 harmonized text of USP ⟨621⟩ (official 1 December 2022) and the EDQM notice for Ph. Eur. 2.2.46; database statements follow the USP Chromatographic Columns and USP–PQRI pages; Fs statements follow Dolan and Snyder (2016) and Stoll (2020); verification and revalidation statements follow 21 CFR 211.194 and ICH Q14. Table 3 uses hypothetical columns and LabVeda calculations. For validated or compendial methods, the applicable procedure and regulatory framework take precedence over the general rules of thumb given here. Evidence review: October 2026.

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