In plain English
A peptide in solution is not only in the solution. Some of it sits on the walls of the vessel, held there by the same mixture of electrostatic and hydrophobic interactions that makes peptides bind to anything else. Adsorption of proteins to solid surfaces has been reviewed as a common but very complicated phenomenon, in which the conformation of the adsorbed molecule matters as much as the amount [1].
Three consequences follow for ordinary laboratory work.
The measured concentration can be lower than the prepared one. A peptide weighed out accurately and dissolved correctly may still arrive at the detector short, because part of it stayed in the tube.
The loss is proportionally worst where it matters most. Walls saturate, so a dilute solution loses a larger fraction than a concentrated one [2].
It is not predictable. In a study of eight iodinated peptides across six container types, charge, size and hydrophobicity gave no pattern that predicted which surface would retrieve which peptide [3].
The short version
- Dilute peptide solutions can lose most of their peptide to container walls [2].
- Loss grows with surface area per unit volume and falls with concentration [2].
- No property of the peptide reliably predicts which surface is best [3].
Key facts
| Observation | What was measured |
|---|---|
| Up to 90% or more lost | Mastoparan X, melittin and magainin 2 at typical working concentrations, quantified by analytical HPLC [2] |
| Glass type alone changed yield from 63% to 20% | Iodinated ghrelin, borosilicate versus flint glass [3] |
| Plastic type changed yield from 16% to 86% | Iodinated sulfated cholecystokinin-8, polystyrene versus polypropylene [3] |
| Adsorption follows a Langmuir isotherm | A decapeptide at 0.2–4 µg/mL, plateau near 0.4 µg/cm² [4] |
| Surface ranking for that decapeptide | glass > polypropylene = polyethylene > poly(tetrafluoroethylene) [4] |
| About 20% lost in eight pipetting steps | Heptapeptide toxins through polypropylene tips; 25–40% when acidified [5] |
| Siliconising usually made things worse | Yield improved for only two of eight peptides tested [3] |
| Accuracy is measured with known added amounts | ICH Q2(R2) bases accuracy on the percentage of a known added amount that the method finds [8] |
What the measurements show
Most of the peptide can be gone
The clearest single dataset comes from a 2015 study that used analytical HPLC to quantify how much of three cationic membrane-active peptides — mastoparan X, melittin and magainin 2 — remained in solution after incubation in borosilicate glass vials, ordinary polypropylene tubes and low-binding tubes. At typical experimental concentrations, 90% or more of the peptides could be lost from solution through rapid adsorption to the container walls [2].
Two systematic dependencies came out of the same work, and both are useful at the bench.
- Concentration. Relatively more peptide adsorbed at low concentrations than at high ones, which the authors interpreted as the walls becoming saturated. The practical implication is to keep stock solutions concentrated; their own stocks were handled at 100 µM or above in low-binding tubes [2].
- Surface-area-to-volume ratio. Relatively less peptide adsorbed when the surface area per unit volume was low. A given amount of solution loses less in one larger aliquot than spread thinly across several small ones [2].
The low-binding tubes showed less adsorption than either the borosilicate glass or the ordinary polypropylene, which the authors offered as a practical measure rather than a complete solution [2].
The surface matters, and not in the way intuition suggests
A 2011 study took a different approach: eight iodinated endocrine peptides, incubated for 48 hours in six container types covering borosilicate glass, flint glass, polystyrene and polypropylene, with yield measured by gamma counting [3].
The results are a warning against generalising.
- The idea that all glass is equivalent did not survive. For iodinated ghrelin, yield was 63% from one glass type and 20% from the other [3].
- Plastic differed just as much. For iodinated sulfated cholecystokinin-8, yield ranged from 16% in polystyrene to 86% in polypropylene [3].
- Direction reversed between peptides. Plastic gave the best yield for ghrelin and among the worst for two of the other peptides tested [3].
- Three of the peptides were basic, three acidic and two uncharged, and no pattern of binding preference followed from charge, size or regional charge distribution [3].
The authors’ summary is that the appropriate container has to be determined for each peptide, and that their yield figures should not be transferred to peptides they did not test [3].
A 2004 study of a decapeptide at 0.2–4 µg/mL in water put numbers on the same effect from the analytical side. Poor and variable HPLC response at low concentrations was traced by rinsing experiments to adsorption on the vial surfaces. The adsorption followed a Langmuir isotherm with a plateau near 0.4 µg/cm², and the surface ranking was glass > polypropylene = polyethylene > poly(tetrafluoroethylene) — with considerable differences between glass vials from different suppliers [4].
Pipette tips count as labware
Containers are the obvious suspect, but transfers add their own losses. In a study of ten heptapeptide toxin congeners handled with glass and polypropylene pipettes and vials, about 20% of the analyte was lost to polypropylene tips after eight successive pipetting steps, rising to 25–40% when the solutions were acidified. Glass tips lost up to 30%, but only from acidified aqueous solutions, and the losses depended on the solvent composition and on which congener was present [5].
The lesson is arithmetical: adsorption is counted per surface contacted, not per container, so a procedure with many transfers has many opportunities to lose material.
What reduces the loss
Published measures fall into four groups. None of them is universal, and two of them contradict each other across studies, which is itself the main finding.
| Measure | What was reported |
|---|---|
| Low-binding containers | Less adsorption of three cationic peptides than in borosilicate glass or ordinary polypropylene [2]. In an LC-MS study of 50 peptides from a digested model protein, a modified polypropylene gave yields above 80% over 24 hours during storage and preparation [6] |
| Keep concentrations high | Proportional loss falls as concentration rises, because walls saturate; stocks handled at 100 µM or above [2] |
| Solvent additives | Glycerol to 50%, or Triton X-100 to 0.2 mM, reduced losses of protein to glass and plastic [7]. For a recombinant protein, additives ranked Tween 20 > Tween 80 > Pluronic F-127 > Pluronic F-68, with Tween 20 most effective on glass and polypropylene vials [9] |
| Carrier protein | Adding bovine serum albumin improved yield of the iodinated peptides, and lyophilising peptide plus albumin in the best-suited tube gave more than 89% yield for all eight [3]. The earlier protein study reached the opposite conclusion: coating surfaces with albumin was less effective than modifying the solvent, and adding albumin to the solvent was counterproductive [7] |
| Organic content in the solvent | Adjusting the methanol fraction alleviated losses of the heptapeptide congeners, but the fraction needed differed between congeners [5] |
Two measures deserve a caution rather than a recommendation.
Siliconising is not a default. Siliconising glassware has long been suggested, but in the eight-peptide study it improved yield for only two of the eight, and losses increased for the rest [3].
Presaturating a container can backfire. Pre-conditioning walls with the peptide reduces subsequent adsorption, but adsorption to borosilicate glass and polypropylene was shown to be at least partly reversible. A presaturated wall can act as a reservoir that releases peptide back into solution during a later step, giving a higher concentration than intended [2].
Ionic strength was tested in the same work and did not help: there was at most a slight trend with sodium chloride concentration in glass vials, and no clear dependence in polypropylene [2].
Adsorption inside the analytical system
The problem does not stop at the sample vial. A review of strategies to reduce aspecific adsorption in LC-MS bioanalysis describes nonspecific binding at pipette tips, sample tubes and vials, and separately at the autosampler, the column and the mass spectrometer, where it shows up as carryover — analyte from one run appearing in the next. Carryover biases both identification and quantification, and linearity, sensitivity and repeatability all suffer. The review’s blunt conclusion is that the diversity of peptide and protein properties means there is no general approach, so the strategies have to be applied and tested case by case [10].
A 2022 study worked through that systematically with 50 peptides obtained by digesting a model protein. Adsorption correlated significantly with the peptides’ hydrophobicity index and chain length (p < 0.05), but those parameters explained the behaviour of only about 30% of the peptides. Having classified the peptides by response and adsorption, the authors optimised container choice and the chromatographic conditions — a C8 column, a higher flow rate of 0.4 mL/min, a shallower gradient and a stronger needle-wash solution — and reduced carryover for the worst-affected peptides to roughly one part in 150 [6]. How a chromatogram records such effects is covered in Reading an HPLC Chromatogram.
Two practical notes follow. Carryover is measured, not assumed: a blank run after a high-concentration sample is the test. And a method’s accuracy, which ICH Q2(R2) bases on the percentage of a known added amount of analyte that the method finds, already folds adsorption losses into the number, provided that experiment uses the same containers and transfers as the real samples [8]. Method characteristics are discussed in Analytical Method Validation.
How to tell adsorption from something else
A low result has several possible causes, and they are distinguishable.
| Observation | Adsorption | Alternative explanation |
|---|---|---|
| Yield falls as the solution is diluted | Characteristic: walls take a fixed areal amount, so dilute solutions lose a larger share [2, 4] | Dilution error, or a detection limit problem |
| Rinsing the empty vessel with a strong solvent returns analyte | Direct evidence of material on the wall [4] | — |
| A fresh container of a different material changes the result | Surface-dependent, as in the glass and plastic comparisons [3, 4] | — |
| Loss grows with the number of transfers | Each contacted surface contributes [5] | — |
| Material is lost but a visible haze appears | — | Aggregation or precipitation; see Peptide Aggregation |
| Loss grows with time at temperature, with new peaks | — | Chemical degradation; see Peptide Storage and Stability |
| Nothing dissolved properly to begin with | — | Solubility; see Predicting Peptide Solubility |
A worked check on the arithmetic shows why dilute work is where this bites. The reported Langmuir plateau for a decapeptide was about 0.4 µg/cm² [4]. A small tube holding 1 mL of solution presents a wetted surface of several square centimetres, so a saturated wall can hold on the order of a microgram. That is negligible against a 1 mg/mL stock and most of the material in a 1 µg/mL working solution. Unit conversions of this kind are set out in Units and Calculations in the Lab.
General laboratory practice
Drawn from the measures above, and framed as what to establish rather than what to assume.
- Determine the container for the peptide in hand. Yield differs between glass types and between plastic types, and the ranking is not transferable between peptides [3, 4].
- Keep stocks concentrated and dilute late. Proportional loss is lowest at high concentration [2].
- Minimise contacted surface. Fewer transfers, fewer small aliquots, and a lower surface-area-to-volume ratio where the design allows [2, 5].
- Check additives rather than assuming them. Glycerol, a non-ionic surfactant or a carrier protein may help, and a carrier protein has been found both helpful and counterproductive in different systems; whichever is chosen must be compatible with the analysis that follows [3, 7, 9].
- Measure yield in the real containers. A yield figure obtained in different labware does not describe the method as run [8, 10].
- Check carryover deliberately. A blank immediately after the highest-concentration sample is the standard test [6, 10].
What this means in Australia
- Adsorption is a method-validation issue, not a product issue. A low measured concentration may reflect the containers and transfers used, so yield has to be established within the method as performed. ICH Q2(R2) defines accuracy as mean percent yield of a known added amount [8].
- Accredited testing. The National Association of Testing Authorities (NATA) accredits Australian laboratories to ISO/IEC 17025, the international standard for the competence of testing and calibration laboratories [11]. Yield and carryover are the kind of method characteristics such accreditation covers.
- Legal status is unaffected. Status under the current Poisons Standard (October 2026) attaches to the substance named there; several peptides are listed in Schedule 4 (prescription only) and Appendix D, clause 5 (possession without authority is illegal) [12]. Handling practice does not alter that.
- Labels do not change status. The TGA has stated that a “research use only” disclaimer does not make supply lawful, and does not change a product’s regulatory status, permit importation, or remove advertising or supply obligations [13].
This is general information, not legal advice. Laws change; check the current Poisons Standard and TGA guidance.
Frequently asked questions
Why do peptides stick to tubes?
Because peptide surfaces carry both charged and hydrophobic groups, which interact with glass and polymer surfaces. Adsorption of proteins and peptides to solid surfaces is a well-described and complicated phenomenon in which the adsorbed molecule’s conformation also changes [1].
How much peptide can be lost?
In one HPLC study, 90% or more of three cationic peptides was lost from solution at typical working concentrations [2]. Yields between 16% and 86% have been reported for a single peptide depending only on the plastic used [3].
Does glass or plastic adsorb more?
It depends on the peptide. For one decapeptide the ranking was glass > polypropylene = polyethylene > poly(tetrafluoroethylene) [4], while in an eight-peptide comparison some peptides were retrieved better from plastic and others from glass, with no predictive pattern [3].
Do low-binding tubes help?
They reduced adsorption of three cationic peptides compared with borosilicate glass and ordinary polypropylene [2], and a modified polypropylene gave yields above 80% over 24 hours for peptides prone to adsorption [6]. They reduce the problem rather than remove it.
Does siliconising glassware help?
Not reliably. In the eight-peptide study, siliconising improved yield for only two peptides and increased losses for the other six [3].
Should albumin be added to the solution?
The evidence is divided. Adding bovine serum albumin improved yield of iodinated peptides, and lyophilising peptide with albumin gave more than 89% yield [3], while an earlier protein study found adding albumin to the solvent counterproductive compared with modifying the solvent itself [7]. It also adds a large protein to every subsequent analysis.
How is adsorption distinguished from degradation?
Adsorption is surface-dependent and concentration-dependent, and rinsing the empty vessel retrieves analyte [2, 4]. Degradation produces new peaks and worsens with time and temperature rather than with container material.
References
- Nakanishi K, Sakiyama T, Imamura K. On the adsorption of proteins on solid surfaces, a common but very complicated phenomenon. J Biosci Bioeng. 2001;91(3):233–244. doi:10.1263/jbb.91.233. PMID: 16232982
- Kristensen K, Henriksen JR, Andresen TL. Adsorption of cationic peptides to solid surfaces of glass and plastic. PLoS One. 2015;10(5):e0122419. doi:10.1371/journal.pone.0122419. PMID: 25932639
- Goebel-Stengel M, Stengel A, Taché Y, Reeve JR Jr. The importance of using the optimal plasticware and glassware in studies involving peptides. Anal Biochem. 2011;414(1):38–46. doi:10.1016/j.ab.2011.02.009. PMID: 21315060
- Grohganz H, Rischer M, Brandl M. Adsorption of the decapeptide Cetrorelix depends both on the composition of dissolution medium and the type of solid surface. Eur J Pharm Sci. 2004;21(2–3):191–196. doi:10.1016/j.ejps.2003.10.008. PMID: 14757490
- Altaner S, Puddick J, Wood SA, Dietrich DR. Adsorption of ten microcystin congeners to common laboratory-ware is solvent and surface dependent. Toxins (Basel). 2017;9(4):129. doi:10.3390/toxins9040129. PMID: 28383495
- Zhang Y, Yang J, Ma Y, Cao L, Huang Q. Nonspecific adsorption evaluation and general minimization strategy in peptide analysis based on ultra-performance liquid chromatography-mass spectrometry. Se Pu. 2022;40(7):616–624. doi:10.3724/SP.J.1123.2021.12012. PMID: 35791600
- Suelter CH, DeLuca M. How to prevent losses of protein by adsorption to glass and plastic. Anal Biochem. 1983;135(1):112–119. doi:10.1016/0003-2697(83)90738-8. PMID: 6670734
- International Council for Harmonisation. ICH Q2(R2): Validation of analytical procedures. 2023. database.ich.org. Accessed 25 October 2026.
- Johnston TP. Adsorption of recombinant human granulocyte colony stimulating factor (rhG-CSF) to polyvinyl chloride, polypropylene, and glass: effect of solvent additives. PDA J Pharm Sci Technol. 1996;50(4):238–245. PMID: 8810839
- Maes K, Smolders I, Michotte Y, Van Eeckhaut A. Strategies to reduce aspecific adsorption of peptides and proteins in liquid chromatography-mass spectrometry based bioanalyses: an overview. J Chromatogr A. 2014;1358:1–13. doi:10.1016/j.chroma.2014.06.072. PMID: 25022477
- National Association of Testing Authorities, Australia (NATA). Accreditation programs, including ISO/IEC 17025 testing and calibration. nata.com.au. Accessed 25 October 2026.
- Therapeutic Goods (Poisons Standard—October 2026) Instrument 2026 (F2026L01327). Federal Register of Legislation. legislation.gov.au/F2026L01327/latest/text. Accessed 25 October 2026.
- Therapeutic Goods Administration. Understanding your responsibilities when importing, compounding and supplying unapproved peptide products. Safety advisory, 13 April 2026. tga.gov.au. Accessed 25 October 2026.
