Peptide Calculator Guide: How to Work Out Concentration, Mass and Volume for Lyophilised Peptides
How a peptide calculator actually works, and how to do the same maths by hand: mass in the vial, solvent volume, the mg/mL you get, converting to µg/µL and molarity, working backwards from a target concentration to the volume of solvent to add, dilution series, and the mistakes that make two calculators give different answers. Written for laboratory preparation of research peptides.
Every peptide calculator on the internet, including ours, does one piece of arithmetic: it divides the mass of peptide in a vial by the volume of solvent added, and then dresses the result up in whatever units the reader asked for. That is worth saying at the start, because a search for "peptide calculator" returns dozens of tools that look different, ask for different inputs, and sometimes give different answers, and the differences are almost never in the maths. They are in what was typed in. This guide explains the calculation from first principles so that a researcher can do it by hand, check any calculator against it, choose a concentration that suits the assay, work backwards to the volume of solvent to add, build a dilution series, and recognise the handful of input mistakes that account for nearly every disagreement between tools. It is written for laboratory preparation of research peptides. It does not describe administration of anything to anyone.
The one equation
concentration (mg/mL) = peptide mass (mg) / solvent volume (mL)
A 5 mg vial made up with 2 mL of solvent is a 2.5 mg/mL solution. A 10 mg vial made up with 2 mL is 5 mg/mL. The vial label gives the mass; the pipette gives the volume; the division gives the concentration. Everything else in this guide is a rearrangement of that line or a unit conversion applied to its answer. Our reconstitution calculator at /calculator performs exactly this division and shows the result in mg/mL and µg/mL, which is all a "peptide calculator" is.
Two things about the inputs deserve care before any calculation. The mass on the label is the nominal quantity of lyophilised material, and depending on the supplier it may be the gross powder mass or the net peptide mass; the section on net peptide content below explains why that matters. The volume is whatever was actually delivered into the vial, which is not the same as the volume on the bottle of solvent or the number a calculator suggested; it is the number read from a calibrated pipette. If either input is approximate, the output is approximate to the same degree, however many decimal places the calculator displays.
Working backwards: from a target concentration to a solvent volume
In practice a researcher rarely starts with a volume and asks what concentration it gives. The assay has a working concentration, the stock needs to be some convenient multiple of it, and the question is how much solvent to add to hit that stock concentration. Rearranging the equation gives the answer.
solvent volume (mL) = peptide mass (mg) / target concentration (mg/mL)
Solvent volume for a target stock concentration (arithmetic only; volumes above the vial capacity need a larger vessel) Peptide mass 1 mg/mL 2 mg/mL 5 mg/mL 10 mg/mL 2 mg 2.0 mL 1.0 mL 0.4 mL 0.2 mL 5 mg 5.0 mL 2.5 mL 1.0 mL 0.5 mL 10 mg 10.0 mL 5.0 mL 2.0 mL 1.0 mL 20 mg 20.0 mL 10.0 mL 4.0 mL 2.0 mL 50 mg 50.0 mL 25.0 mL 10.0 mL 5.0 mL
Two constraints sit on top of the arithmetic. The first is the vial: a small research vial, commonly around 3 mL, will not take 5 mL of solvent, so a 5 mg vial cannot be made up to 1 mg/mL in its own container and either needs transferring to a larger vessel or a higher stock concentration. The second is solubility: some peptides will not dissolve at 10 mg/mL in water at all and need a different solvent or a lower concentration, our solubility calculator suggests a starting solvent from the sequence, though it does not predict a solubility limit. The bacteriostatic water guide covers when bacteriostatic water is the right solvent and when sterile water, dilute acetic acid or a small proportion of DMSO is needed instead.
Unit conversions that trip people up
Peptide work moves between milligrams, micrograms, millilitres and microlitres constantly, and most calculator disagreements are unit slips rather than arithmetic errors. The prefixes are fixed by the SI: milli is one thousandth, micro is one millionth, so there are a thousand micrograms in a milligram and a thousand microlitres in a millilitre. The useful consequence is that mg/mL and µg/µL are the same number, because both the top and the bottom of the fraction have been divided by a thousand. A 2.5 mg/mL solution is a 2.5 µg/µL solution, and a 10 µL aliquot of it contains 25 µg.
Equivalent expressions of the same concentration mg/mL µg/µL µg per 50 µL µg per 10 µL g/L 0.5 0.5 25 5 0.5 1 1 50 10 1 2 2 100 20 2 2.5 2.5 125 25 2.5 5 5 250 50 5 10 10 500 100 10
The column that matters for planning an experiment is usually the mass in a given aliquot volume: how many micrograms are in the 10 µL, 25 µL or 50 µL that a pipette will deliver into a well or a tube. That is concentration multiplied by volume, with the units made to match. At 2 mg/mL, a 25 µL aliquot holds 2 µg/µL × 25 µL = 50 µg.
From mg/mL to molarity
Many assays are specified in molar terms, micromolar or nanomolar, because what matters biochemically is the number of molecules rather than their mass. Converting requires the molecular weight of the peptide in grams per mole, which is on the COA or can be computed from the sequence with our molecular weight calculator. Concentration in g/L is numerically identical to mg/mL, so the conversion is one division.
molarity (mol/L) = concentration (g/L) / molecular weight (g/mol); × 1,000 for mM, × 1,000,000 for µM
Worked example: a hypothetical peptide with molecular weight 1,250 g/mol made up to 2 mg/mL. Two grams per litre divided by 1,250 grams per mole is 0.0016 mol/L, which is 1.6 mM or 1,600 µM. To make a 10 µM working solution from that stock is a 160-fold dilution: 6.25 µL of stock made up to 1 mL with buffer, or more practically an intermediate 100 µM solution first. Our molarity calculator does this conversion from mass, volume and molecular weight, and is the tool to use whenever an assay protocol is written in molar units.
One trap: the molecular weight should be that of the peptide as supplied, including any counter-ion, if the mass on the label is the mass of the salt. Each acetate counter-ion adds about 60 g/mol and each trifluoroacetate about 114 g/mol, so for a small peptide with several basic residues the salt can weigh from around 5 per cent to more than 20 per cent more than the free peptide, and using the free-peptide value against a salt mass overstates the molarity by that margin. The COA should state which form the mass refers to; if it does not, the net peptide content figure discussed next usually resolves it.
Net peptide content: why the label mass is not the peptide mass
Lyophilised peptide powder is not pure peptide. It contains bound water, counter-ions from purification and sometimes residual salts, so the peptide itself is commonly quoted by manufacturers as 60 to 90 per cent of the powder by mass, depending on the sequence and the counter-ion. Purity, the figure on an HPLC report, is a different quantity: it describes what fraction of the peptide-related material is the target sequence rather than truncated or modified sequences, and says nothing about the water and salt. A vial can be 99 per cent pure by HPLC and 80 per cent peptide by mass at the same time. Our HPLC report guide explains how the two figures are derived and why a purity number on its own does not tell you how much peptide is in the vial.
Where only relative concentrations matter, for example comparing dilutions of one stock, the label mass is often used as it stands, with the understanding that the true peptide concentration is lower than calculated, sometimes by 10 to 40 per cent. Where the assay is sensitive to absolute concentration, the correction is straightforward: multiply the label mass by the net peptide content before dividing by volume. A 5 mg vial with 82 per cent net peptide content made up to 2 mL is 5 × 0.82 / 2 = 2.05 mg/mL of peptide, not 2.5. Where a supplier states net peptide content on the COA, that figure should be used; where it does not, the content is unknown rather than assumable, and the concentration should be recorded as nominal (by label mass) or the content measured by amino acid or elemental analysis.
Building a dilution series
A stock solution is usually stronger than any working concentration, and the working concentrations are made by dilution. The relationship is the same equation once more, applied to the stock rather than the powder: the mass of peptide is conserved, so concentration times volume before dilution equals concentration times volume after it.
C1 × V1 = C2 × V2, so stock volume needed V1 = (C2 × V2) / C1
Decide the final working concentration and the volume needed for the experiment, including a margin for pipetting losses and replicates. Choose an intermediate concentration if the direct dilution would require a stock volume below about 2 µL, which is where pipetting error becomes large. A ten-fold intermediate step is the usual fix. Calculate V1 for each step, pipette the stock into the diluent (not the other way round, to avoid concentrated peptide sitting on the tube wall), and mix by gentle inversion. Label each tube with concentration, date and the stock it came from. Record the actual volumes pipetted, not the planned ones, if they differ. Treat diluted solutions as less stable than the stock; the storage protocol explains why dilute peptide solutions adsorb to plastic and degrade faster. Example: a 2 mg/mL stock of the 1,250 g/mol peptide above is 1.6 mM. To make 1 mL of 1 µM working solution directly would need 0.625 µL of stock, which no ordinary pipette delivers reliably. Instead, 10 µL of stock into 1,590 µL of buffer gives 10 µM; then 100 µL of that into 900 µL gives 1 µM. Two steps, each with a pipette used inside its accurate range: a P10 or P20 for the stock, a P200 and a P1000 for the rest.
Why calculators disagree, and how to check one
When two peptide calculators give different numbers for what looks like the same problem, the cause is nearly always one of the following. They were given different masses, because one used the label figure and the other a net-peptide-corrected figure. They were given different volumes, because one included a solvent volume that was actually in the vial and the other the nominal volume of the solvent container. One of them reports a device-specific graduation rather than a concentration, which is not a laboratory unit and is outside this guide. Or one rounds at an intermediate step, which produces small discrepancies in the last decimal place that are harmless but alarming. The arithmetic itself, a single division, is not something a working calculator gets wrong.
Checking a calculator by hand Input Value Hand calculation What the tool should show Vial mass 5 mg Solvent volume 2 mL 5 / 2 2.5 mg/mL Same, as µg/µL no change 2.5 µg/µL Mass in 10 µL 2.5 × 10 25 µg Molarity at MW 1,250 2.5 / 1250 × 1000 2.0 mM With 80% net content 5 × 0.8 / 2 2.0 mg/mL
Any calculator that reproduces the rows it covers is doing the maths correctly. Our reconstitution calculator gives the concentration row and our molarity calculator the molarity row; the aliquot and net-content rows are hand calculations. If a tool disagrees, the mismatch is an input or a unit, and the fix is to find which. The embeddable version of our reconstitution calculator at /free-peptide-calculator is the same code, offered so that other laboratory sites can carry the tool without writing their own.
Measuring the volume properly
The calculation is exact; the measurement is not. A concentration is only as good as the volume that went into the vial, and the volume is only as good as the instrument that delivered it. A calibrated air-displacement pipette within ISO 8655 limits is typically accurate to around one per cent at its nominal volume for pipettes of 10 µL and above, less accurate at the bottom of its range and for the smallest pipettes, and should be used with a fresh tip, pre-wetted, held vertically, with the liquid dispensed slowly against the vial wall. The printed markings on a vial or a solvent container are not calibrated and should not be used to measure a volume. For solvent volumes above 1 mL, a serological pipette or a 1,000 µL pipette used in repeated aliquots is the usual choice. The reconstitution guide covers the handling steps: solvent added down the wall of the vial rather than onto the powder, swirling rather than shaking, and time allowed for full dissolution before any concentration is recorded as real.
The five laboratory calculators and what each one is for
Reconstitution calculator (/calculator): mass and volume to mg/mL and µg/mL; the general-purpose peptide calculator. Molarity calculator (/peptide-molarity-calculator): mass, volume and molecular weight to mM and µM. Molecular weight calculator (/peptide-molecular-weight-calculator): amino-acid sequence to average molecular weight, formula and net charge, for peptides whose COA does not state a molecular weight. Solubility calculator (/peptide-solubility-calculator): sequence to net charge and hydropathy, with a suggested first solvent, to avoid attempting a 10 mg/mL aqueous stock of a peptide that will not dissolve in water. Embeddable reconstitution calculator (/free-peptide-calculator): the reconstitution tool as a free embed for laboratory websites. All five are laboratory tools for preparing research peptides for in-vitro research. The products in our catalogue are supplied for laboratory research use only. They are not licensed medicines and are not supplied, presented or intended for human or veterinary use. Whether any product falls within medicines law depends on what it is and how it is presented and supplied, which is why nothing on this page or in the calculators describes or supports administration of any substance to a person or an animal. The maths of concentration is the same in any laboratory, and that is all it is.
Frequently Asked Questions How does a peptide calculator work? It divides the mass of peptide in the vial by the volume of solvent added to give a concentration in mg/mL, then converts that into whatever unit the researcher needs: µg/µL, µg per 100 µL, or molarity if the molecular weight is known. Every peptide calculator is doing that one division; the differences are in the units it accepts and displays.
How much bacteriostatic water do you add to a peptide vial? There is no fixed volume: it follows from the stock concentration the experiment needs, not the other way round. Volume (mL) equals peptide mass (mg) divided by target stock concentration (mg/mL), so 5 mg at 2 mg/mL is 2.5 mL and 10 mg at 5 mg/mL is 2 mL. The choice is set by the assay, the solubility of the peptide and the capacity of the vial, and bacteriostatic water is not always the right solvent; see our bacteriostatic water guide. This is laboratory arithmetic for in-vitro work only.
What is the difference between mg/mL and µg/µL? None. One milligram per millilitre is the same concentration as one microgram per microlitre, because both numerator and denominator have been divided by a thousand. Calculators that show both are showing the same number twice.
How do you convert a peptide concentration to molarity? Divide the concentration in g/L (which is numerically the same as mg/mL) by the molecular weight in g/mol to get mol/L, then multiply by 1,000 for mM or by 1,000,000 for µM. A 2 mg/mL solution of a hypothetical peptide with molecular weight 1,250 g/mol is 1.6 mM.
Why do two peptide calculators give different answers? Usually because one is using the label mass and the other is using net peptide content, or because one rounds intermediate values. The underlying arithmetic is identical; if two tools disagree, one of them has been given a different input.
What does net peptide content mean and does it change the calculation? Lyophilised peptide powder contains water and counter-ions as well as the peptide itself, so the peptide is commonly quoted by manufacturers as 60 to 90 per cent of the powder mass, depending on the sequence and the counter-ion. If the COA gives net peptide content, multiply the label mass by it before dividing by volume; otherwise the calculated concentration is that of the powder, not the peptide.
How accurate does the volume measurement need to be? The concentration is only as accurate as the volume. A calibrated piston pipette within ISO 8655 limits is typically accurate to around one per cent at its nominal volume for pipettes of 10 µL and above, and less accurate at the bottom of its range; the printed markings on a vial or a solvent container are not calibrated at all. For a 2 mL make-up, a 0.1 mL error in volume is roughly a five per cent error in concentration before any assay begins.
Can this calculator be used for personal use? No. The calculators and this guide are tools for preparing research peptides in a laboratory for in-vitro research. The products are supplied for laboratory research use only, are not licensed medicines, are not for human or veterinary use, and nothing here describes administration to a person or an animal. Questions about any medicine belong with a prescriber or pharmacist.
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For in-vitro laboratory research use only. Not for human or veterinary use, consumption, or therapeutic application. No medical claims are made.
